Composite exosome for encapsulating linear macromolecular nanoparticles as well as preparation method and application of composite exosome

Abstract

The invention discloses a composite exosome encapsulating linear macromolecular nanoparticles as well as a preparation method and application thereof, and belongs to the technical field of biological materials and regenerative medicine. Aiming at the pain points that in the prior art, when linear macromolecules such as collagen and hyaluronic acid are encapsulated by exosomes, the encapsulation efficiency is low, the structure of the exosomes is easy to damage, and the molecular weight adaptability is poor, the invention provides the preparation method which comprises the following steps: firstly, pretreating the collagen of 5 kDa to 330 kDa and the hyaluronic acid of 10 kDa to 2200 kDa into dense nanoparticles of 10 to 150 nm and PDI less than or equal to 0.15 through a nanoprecipitation method, an electrostatic complexation method and the like; and encapsulating in the exosome through an electroporation method, a freeze-thaw cycle method or an ultrasonic method, and purifying to obtain the composite exosome. According to the method, linear macromolecules with different molecular weights are adapted by flexibly adjusting process parameters, the encapsulation efficiency is remarkably improved, and the membrane structure of the exosome can be kept complete. The obtained composite exosome has the signal transduction function of the exosome and the biological activity of linear macromolecules, and generates a synergistic effect in tissue repair.

Description

Technical Field

[0001] This invention belongs to the field of biomaterials and regenerative medicine technology, specifically relating to a composite exosome encapsulating linear macromolecular nanoparticles, its preparation method, and its application. Background Technology

[0002] Exosomes are nanoscale vesicles (<200 nm in diameter) secreted by cells. They have a lipid bilayer structure and naturally carry bioactive molecules such as proteins and nucleic acids, playing a key role in intercellular communication. Due to their low immunogenicity, good biocompatibility, and targeting properties, exosomes are considered to be highly promising natural drug delivery carriers.

[0003] However, using exosomes as carriers to encapsulate exogenous macromolecules, especially linear macromolecules, faces significant challenges. Collagen and hyaluronic acid are typical linear, flexible long-chain polymers with a hydrodynamic radius ≥200 nm in their untreated state, which is larger than the internal cavity of exosomes (<200 nm). If traditional methods such as electroporation and freeze-thaw methods are used for encapsulation, the following problems exist: (1) Extremely low encapsulation efficiency: Long-chain structures are difficult to effectively enter through the transient pores of the exosome membrane, and the encapsulation rate of existing technologies is usually less than 10% (as shown in Examples 9 and 10); (2) Damage to exosome structure: Forced encapsulation leads to a large number of exosomes losing their activity due to membrane structure rupture; (3) Component aggregation: Linear macromolecules entangle and aggregate with each other outside the exosome, resulting in a product particle size distribution polydispersity (PDI) >0.3, which cannot meet the uniformity requirements.

[0004] In existing technologies, related research only focuses on optimizing exosome encapsulation methods (such as adjusting electroporation parameters and improving purification processes), failing to recognize that the fundamental reason for low encapsulation efficiency is the "mismatch between linear macromolecular structures and exosome cavities." Furthermore, the impact of molecular weight differences on encapsulation effectiveness has been ignored. Existing technologies have not actively enhanced the interaction between linear macromolecular nanoparticles and exosomes by modifying their surface charge, thus failing to address the issues of encapsulation efficiency and stability. Low molecular weight (≤10 kDa) linear macromolecules may pass through membrane pores but are prone to leakage, while high molecular weight (≥100 kDa) linear macromolecules are more difficult to enter exosomes and easily cause membrane damage, and medium molecular weight (10-100 kDa) macromolecules pose a risk of aggregation. The lack of a universal encapsulation strategy adaptable to linear macromolecules of different molecular weights further limits the application range of exosome carriers. The core contradiction lies in the mismatch between the hydrodynamic radius of linear macromolecules and the internal cavity size of exosomes, the physical shear damage to the lipid bilayer caused by long-chain molecules, and the compatibility issues arising from molecular weight differences. Existing technologies have not proposed effective solutions to these contradictions.

[0005] Therefore, developing a new exosome encapsulation engineering strategy has become an urgent problem to be solved. Summary of the Invention

[0006] Therefore, the purpose of this invention is to provide a composite exosome encapsulating linear macromolecular nanoparticles, its preparation method, and its application. It utilizes physical and chemical methods to "reshape" linear macromolecules into nanoparticles, solving the problem of mismatch between their structure and membrane characteristics with the internal space of exosomes, significantly improving encapsulation efficiency, and is applicable to linear macromolecules of different molecular weights.

[0007] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a composite exosome encapsulating linear macromolecular nanoparticles, wherein the exosome membrane is encapsulated with long-chain polymer nanoparticles, the membrane integrity being ≥90%; the hydrodynamic diameter of the long-chain polymer nanoparticles is between 10 nm and 150 nm, the polymer dispersion is ≤0.15, and the surface is positively charged.

[0008] Among them, exosome membrane integrity refers to the proportion of exosome vesicles that retain an intact membrane structure after encapsulation.

[0009] The exosomes are derived from various cells or gene-recombined cells of human, animal or plant origin.

[0010] Polymer Dispersion (PDI) is abbreviated as PDI.

[0011] The nanoparticles have a positively charged surface to enhance their subsequent binding efficiency with the exosome membrane.

[0012] Based on the above technical solution, the long-chain polymer is further selected from one or more of polylactic acid and its derivatives, polycaprolactone, polydeoxyribonucleotides, polynucleotides, chitosan and its derivatives, silk fibroin, collagen and hyaluronic acid.

[0013] Based on the above technical solution, the long-chain polymer is collagen and / or hyaluronic acid, wherein the molecular weight of the collagen is 5 kDa-330 kDa, and the molecular weight of the hyaluronic acid is 10 kDa-2200 kDa.

[0014] Among them, collagen with a molecular weight of 5-10 kDa is considered low molecular weight; When the molecular weight of collagen is 10-100 kDa, it is considered a medium molecular weight. Collagen with a molecular weight of 100-330 kDa is considered high molecular weight.

[0015] Among them, hyaluronic acid with a molecular weight of 10-50 kDa is considered a low molecular weight. When the molecular weight of hyaluronic acid is 50-500 kDa, it is considered a medium molecular weight. Hyaluronic acid with a molecular weight of 500-2200 kDa is considered high molecular weight.

[0016] Secondly, the present invention provides a method for preparing a composite exosome encapsulating linear macromolecular nanoparticles, comprising the following steps: 1) Prepare long-chain polymer nanoparticles using physical or chemical methods; 2) Mix nanoparticles and exosomes at a mass ratio of 1:10-10:1 in a buffer solution with pH 7.2-7.4 and incubate for 10-60 minutes at a mixing temperature of 4-25℃; 3) Nanoparticles are encapsulated inside exosomes using electroporation, freeze-thaw cycles, or ultrasound. 4) Purify the exosomes by ultracentrifugation or size exclusion chromatography to remove free nanoparticles and obtain composite exosomes.

[0017] In step 2), the preferred mass ratio of nanoparticles to exosomes is 1:2-2:1, and the preferred buffer solution is phosphate buffer. In step 4), the reaction product is purified by ultracentrifugation or size exclusion chromatography to remove ≥95% of the unencapsulated free nanoparticles and obtain pure composite exosomes; the particle size distribution (PDI) of the purified composite exosomes is ≤0.2 and the membrane integrity is ≥90%.

[0018] Based on the above technical solution, further, in step 1): Physical methods include nanoprecipitation, electrostatic complexation, or thermally induced self-assembly. The chemical method is the cross-linking method; The long-chain polymers are collagen and / or hyaluronic acid. The nanoparticles have a hydrodynamic diameter of 10-150 nm, a PDI ≤ 0.15, and are positively charged by cationic amino acid modification. The molecular weight of collagen is 5 kDa-330 kDa, and the molecular weight of hyaluronic acid is 10 kDa-2200 kDa. Nanoparticles are positively charged by introducing cationic amino acids through blending or surface modification. The cationic amino acids are selected from one or more of lysine, arginine, and histidine.

[0019] Specifically, for low molecular weight (collagen ≤10 kDa, hyaluronic acid ≤50 kDa): in the nanoprecipitation method, the polymer solution concentration can be increased (1-2 mg / mL), or the microfluidic mixing time can be extended to avoid leakage after encapsulation due to excessively small particles (<10 nm); for high molecular weight (collagen ≥100 kDa, hyaluronic acid ≥500 kDa): in the electrostatic complexation method, the proportion of cationic complexing agents (such as lysine hydrochloride) can be increased (volume ratio 1:1.2-1:1.5), or the ultrasonic time can be extended (5-10 minutes) to ensure that the long chains are fully folded to form dense nanoparticles.

[0020] Based on the above technical solution, further, in step 3): Electroporation method: voltage 300-600 V, capacitance 300-800 μF, pulse count 1-3 times, buffer conductivity ≤10 mS / cm; The freeze-thaw cycle method involves freezing in liquid nitrogen for 5-10 minutes, thawing in a 37°C water bath for 10-15 minutes, and repeating 1-5 times. The ultrasonic method uses a power of 50-150 W, operates for 3 seconds, pauses for 5 seconds, and has a total duration of 3-8 minutes. The temperature is controlled below 4℃.

[0021] Based on the above technical solution, further, when the molecular weight of collagen is ≤10 kDa and the molecular weight of hyaluronic acid is ≤50 kDa, nanoparticles are prepared by nanoprecipitation method, the polymer solution concentration is 0.5-5 mg / mL, and the flow rate ratio of polymer solution to poor solvent in microfluidic chip is 1:2-1:5; When the molecular weight of collagen is ≥100 kDa and the molecular weight of hyaluronic acid is ≥500 kDa, nanoparticles are prepared by electrostatic complexation, with a cationic complexing agent volume ratio of 1:0.5-1:3 and an ultrasonication time of 5-30 minutes.

[0022] The preferred flow rate ratio of the polymer solution to the undesirable solvent is 1:2.5-1:3 to obtain nanoparticles with good monodispersity.

[0023] In the preparation of hyaluronic acid nanoparticles by electrostatic complexation, cationic amino acids such as lysine, arginine, and histidine are used as cationic complexing agents. For high molecular weight hyaluronic acid, the volume ratio of cationic amino acids such as lysine, arginine, and histidine to hyaluronic acid can be preferably adjusted to 1:1.2-1:1.5.

[0024] Thirdly, the present invention provides the application of the above-mentioned composite exosomes encapsulating linear macromolecular nanoparticles in the preparation of products for promoting tissue repair and regeneration.

[0025] Based on the above technical solution, the product is a drug, medical device, cosmetic or food, and the tissue repair and regeneration is skin repair, anti-aging, bone and cartilage defect repair or wound healing.

[0026] Based on the above technical solution, the product further includes the aforementioned composite exosome, as well as a pharmaceutically acceptable carrier or a cosmetically acceptable matrix.

[0027] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention utilizes physical and chemical methods to "reshape" linear macromolecules into nanoparticles, solving the problem of mismatch between their structure and membrane characteristics with the internal space of exosomes, significantly improving encapsulation efficiency, and is applicable to linear macromolecules of different molecular weights, resulting in a revolutionary improvement in encapsulation efficiency.

[0028] 2. This invention maintains the integrity of exosomes: it avoids physical damage to the membrane by long-chain molecules, and the integrity rate of the exosome membrane structure after engineering is greatly improved. The integrity (vesicle ratio) of the exosome membrane after encapsulation is ≥90%.

[0029] 3. The composite exosomes prepared in this invention possess both the natural signal transduction function of exosomes (such as carrying miR-125b to promote cell differentiation) and the biological functions of collagen / hyaluronic acid (such as enhancing cell adhesion and replenishing the extracellular matrix). The synergistic effect of these two components significantly enhances the tissue repair rate, producing a synergistic effect of "1+1>2". 4. The process of this invention is controllable and easy to scale up: the preparation method is mild and the steps are clear. The parameters can be flexibly adjusted for linear macromolecules of different molecular weights. The product has a stability of at least 12 months when stored at 2-8℃, making it suitable for standardized production and quality control.

[0030] 5. This invention has a wide molecular weight adaptability: it can efficiently encapsulate 5 kDa-330 kDa collagen and 10 kDa-2200 kDa hyaluronic acid, covering the commonly used molecular weight range, and solves the pain points of existing technologies where low molecular weight is prone to leakage and high molecular weight is difficult to encapsulate.

[0031] 6. Enhanced encapsulation dynamics of the present invention: By introducing cationic amino acid modification, the surface of the nanoparticles becomes positively charged, generating strong electrostatic attraction with the negatively charged exosome membrane. This not only further improves the encapsulation efficiency but also enhances the retention stability of the encapsulated nanoparticles inside the exosome, reducing the risk of leakage. Attached Figure Description

[0032] To more clearly illustrate the embodiments of the present invention, the accompanying drawings involved in the embodiments will be briefly described below.

[0033] Figure 1This is a schematic diagram of the technical route of the present invention. Detailed Implementation

[0034] This invention belongs to the field of biomaterials and regenerative medicine technology, specifically relating to an engineered modification method for drug delivery carriers, particularly to a preparation system in which linear biomacromolecules such as collagen and hyaluronic acid of different molecular weights (low, medium, and high) are pretreated into nanoparticles with controllable size and then encapsulated inside exosomes, and its application in tissue engineering, regenerative medicine, skin repair and anti-aging.

[0035] Existing technologies only focus on optimizing exosome encapsulation methods (such as adjusting electroporation parameters and improving purification processes), without realizing that "the mismatch between the linear macromolecular structure and the exosome cavity" is the root cause of low encapsulation efficiency. This invention proposes for the first time a pretreatment strategy of "first converting linear macromolecules into positively charged nanoparticles before encapsulation," which solves the structure matching problem from the source and is a groundbreaking technical concept.

[0036] This invention achieves a triple effect of "high encapsulation rate + intact exosome structure + synergistic function" by introducing cations to achieve positive surface charge, limiting nanoparticle size (10-150 nm), PDI (≤0.15), and encapsulation process parameters. Existing technologies cannot simultaneously meet these three technical indicators.

[0037] Existing technologies do not consider the encapsulation differences of linear macromolecules with different molecular weights, and the problems of easy leakage of low molecular weights and difficulty in entry of high molecular weights have not been solved. This invention achieves efficient encapsulation of 5 kDa-330 kDa collagen and 10 kDa-2200 kDa hyaluronic acid by flexibly adjusting the pretreatment process parameters, covering the commonly used molecular weight range, and its versatility is significantly better than that of existing technologies.

[0038] This invention not only solves the "size matching" problem, but also actively enhances the binding force between nanoparticles and exosomes through a "charge adaptation" strategy, forming a dual guarantee. This is a groundbreaking concept that has not been mentioned in the prior art.

[0039] This invention addresses the shortcomings of existing technologies for encapsulating linear macromolecules such as collagen and hyaluronic acid using exosomes, including low encapsulation efficiency, easy destruction of exosome structure, and poor molecular weight adaptability. The invention proposes pre-treating 5 kDa-330 kDa collagen and 10 kDa-2200 kDa hyaluronic acid into dense nanoparticles of 10-150 nm with a PDI ≤ 0.15 using nanoprecipitation or electrostatic complexation. These nanoparticles are then encapsulated within exosomes using electroporation, freeze-thaw cycles, or ultrasonication, followed by purification to obtain composite exosomes. This method significantly improves encapsulation efficiency by flexibly adjusting process parameters to suit linear macromolecules of different molecular weights, while maintaining the integrity of the exosome membrane structure. The resulting composite exosomes combine the signal transduction function of exosomes with the biological activity of linear macromolecules, producing a synergistic effect in tissue repair, and exhibit good stability after 12 months of storage at 2-8°C. The process of this invention is controllable and easy to scale up, and can be widely used in the preparation of drugs, medical devices, cosmetics and food related to skin repair, anti-aging, bone and cartilage defect repair and wound healing, and has important industrialization value.

[0040] The present invention will be described in detail below with reference to embodiments. However, the implementation of the present invention is not limited thereto. Obviously, the embodiments described below are only some embodiments of the present invention. For those skilled in the art, other similar embodiments can be obtained without creative effort and all fall within the protection scope of the present invention. Unless otherwise specified, the raw materials involved in the following embodiments can be directly purchased commercially. Example 1

[0041] Medium molecular weight collagen (85 kDa) nanoparticles were prepared and encapsulated using a nanoprecipitation method. (1) Preparation of collagen nanoparticles: Type I collagen (85 kDa, purity ≥95%) and lysine hydrochloride (mass ratio 10:1) were dissolved in 0.1% acetic acid aqueous solution to prepare a 1 mg / mL solution (solvent); PBS (pH 7.4) was used as a poor solvent; using a microfluidic chip (channel size 50 μm), the collagen solution and exosome suspension were rapidly mixed in the chip at a volume ratio of 1:3 (flow rate ratio: collagen solution 1 mL / min, exosome suspension 3 mL / min), and the effluent was collected to obtain a collagen nanoparticle suspension; the average particle size was 85 nm, the PDI was 0.15, and the surface potential was 15 mV as determined by dynamic light scattering (DLS).

[0042] (2) Mixing: Mix the above nanoparticle suspension with human umbilical cord mesenchymal stem cell-derived exosomes at a mass ratio of 1:1 and incubate at 4°C for 45 minutes.

[0043] (3) Encapsulation: Electroporation was performed using an electroporator at a voltage of 500 V, a capacitance of 500 μF, and a pulse count of 1, with the entire process conducted in an ice bath.

[0044] (4) Purification: The mixture after electroporation was precipitated by ultracentrifugation (100,000 g, 70 min), the supernatant was discarded, the precipitate was resuspended in PBS (pH 7.2-7.4), and then freeze-dried under vacuum to obtain the final complex exosomes. Example 2

[0045] Hyaluronic acid nanoparticles were prepared and encapsulated using an electrostatic complexation method.

[0046] (1) Preparation of hyaluronic acid nanoparticles: Hyaluronic acid (molecular weight 50 kDa, purity ≥98%) and lysine hydrochloride were dissolved in water at a concentration of 0.5 mg / mL. Under magnetic stirring, the lysine hydrochloride solution was slowly added dropwise to the hyaluronic acid solution (volume ratio 1:1), and stirring was continued for 30 minutes. Then, the probe was used for ultrasonication (100 W, working for 2 seconds, intermittent for 5 seconds, for a total of 5 minutes). The temperature was controlled at ≤4℃ in an ice bath to avoid thermal damage, reduce particle size, and make it uniform. Finally, hyaluronic acid nanocomposite was obtained. The average particle size was 65 nm, the PDI was 0.14, and the surface potential was 14 mV as determined by DLS.

[0047] (2) Mixing: Mix the nanoparticle suspension with human umbilical cord mesenchymal stem cell-derived exosomes at a mass ratio of 2:1 and incubate at 25°C for 30 minutes.

[0048] (3) Encapsulation: The freeze-thaw cycle method was used, which involved freezing in liquid nitrogen for 8 minutes and thawing in a water bath at 37°C for 12 minutes, and was repeated once.

[0049] (4) Purification: Same as in Example 1, to obtain complex exosomes. Example 3

[0050] Encapsulated collagen and hyaluronic acid composite nanoparticles.

[0051] (1) Preparation of composite nanoparticles: The collagen nanoparticle suspension prepared in Example 1 and the hyaluronic acid nanoparticle suspension prepared in Example 2 were mixed at a mass ratio of 1:1 and stirred at room temperature for 20 minutes to obtain a composite nanoparticle suspension.

[0052] (2) Mixing: Mix the composite nanoparticle suspension with human umbilical cord mesenchymal stem cell-derived exosomes at a mass ratio of 1:2 and incubate at 4°C for 60 minutes.

[0053] (3) Encapsulation: Ultrasonic method, power 80 W, working time 3 seconds / intermittent time 5 seconds, total duration 5 minutes, ice bath conditions.

[0054] (4) Purification: Use size exclusion chromatography column, elution buffer is PBS (pH 7.4), flow rate is 0.5 mL / min, collect the elution peak (corresponding to complex exosomes) at 20-30 minutes, freeze dry under vacuum to obtain complex exosomes. Example 4

[0055] Preparation and encapsulation of low molecular weight collagen (5 kDa) nanoparticles.

[0056] (1) Nanoparticle preparation: 5 kDa collagen (purity ≥95%) and lysine hydrochloride (mass ratio 10:1) were dissolved in 0.1% acetic acid aqueous solution to prepare a 2 mg / mL solution (solvent); human umbilical cord mesenchymal stem cell-derived exosome suspension was used as a poor solvent; a microfluidic chip (channel size 50 μm) was used, and the flow rate ratio was adjusted to 1 mL / min for collagen solution and 2.5 mL / min for exosome suspension. After rapid mixing, the effluent was collected; the average particle size was 35 nm, the PDI was 0.11, and the surface potential was 14 mV as determined by DLS.

[0057] (2) Mixing: The nanoparticle suspension and human umbilical cord mesenchymal stem cell-derived exosomes were mixed at a mass ratio of 1.5:1 and incubated at 4°C for 60 minutes.

[0058] (3) Encapsulation: The electroporation parameters were adjusted to voltage 450 V, capacitance 600 μF, pulse count 1, and ice bath throughout.

[0059] (4) Purification: Purification by ultracentrifugation (100,000 g, 70 minutes), followed by vacuum freeze drying to obtain the final complex exosomes. Example 5

[0060] Preparation and encapsulation of high molecular weight hyaluronic acid (1000 kDa) nanoparticles.

[0061] (1) Nanoparticle preparation: Hyaluronic acid (1000 kDa, purity ≥98%) and lysine hydrochloride were dissolved in water, each with a concentration of 0.5 mg / mL; under magnetic stirring, the lysine hydrochloride solution was slowly added dropwise to the hyaluronic acid solution at a volume ratio of 1:1.5, and stirring was continued for 60 minutes; the probe was used for ultrasonication (100 W, working for 2 seconds / 5 seconds intermittent, for a total of 8 minutes), and the temperature was controlled at ≤4℃ in an ice bath; the average particle size was 120 nm, the PDI was 0.15, and the surface potential was 15 mV as determined by DLS.

[0062] (2) Mixing: The nanoparticle suspension and human umbilical cord mesenchymal stem cell-derived exosomes were mixed at a mass ratio of 2:1 and incubated at 25°C for 45 minutes.

[0063] (3) Encapsulation: Freeze-thaw cycle method (freeze in liquid nitrogen for 10 minutes, thaw at 37°C for 15 minutes, repeat once).

[0064] (4) Purification: Purification by size exclusion chromatography and freeze drying under vacuum to obtain the final complex exosomes. Example 6

[0065] Medium molecular weight collagen (50 kDa) and hyaluronic acid (200 kDa) composite nanoparticles encapsulated.

[0066] (1) Preparation of composite nanoparticles: Collagen nanoparticles: medium molecular weight type I collagen (50 kDa, purity ≥95%) and lysine hydrochloride (mass ratio 10:1), nanoprecipitation method (microfluidic flow rate ratio 1:3), particle size 65 nm, PDI 0.11, surface potential 13 mV. Hyaluronic acid nanoparticles: medium molecular weight hyaluronic acid (200 kDa, purity ≥98%), electrostatic complexation method (lysine hydrochloride volume ratio 1:1.2), particle size 75 nm, PDI 0.15, surface potential 13 mV. The two were mixed at a mass ratio of 1:1 and stirred at room temperature for 30 minutes.

[0067] (2) Mixing and encapsulation: Mix with human umbilical cord mesenchymal stem cell-derived exosomes at a mass ratio of 1:1, incubate at 4°C for 50 minutes, and encapsulate by sonication (power 100 W, total duration 6 minutes).

[0068] (3) Purification: Purification by size exclusion chromatography and freeze drying under vacuum to obtain the final complex exosomes. Example 7

[0069] Medium molecular weight collagen (85 kDa) nanoparticles were prepared and encapsulated (without introducing lysine hydrochloride) using a nanoprecipitation method. Type I collagen (85 kDa, purity ≥95%) was dissolved in 0.1% acetic acid aqueous solution, and other steps and conditions were exactly the same as in Example 1 to prepare composite exosomes. The average particle size of the collagen nanoparticle suspension was 135 nm, PDI was 0.16, and surface potential was -5 mV, as determined by dynamic light scattering (DLS). Example 8

[0070] Encapsulated collagen and hyaluronic acid composite nanoparticles (without introducing lysine hydrochloride): The collagen nanoparticle suspension prepared in Example 7 and the hyaluronic acid nanoparticle suspension prepared in Example 2 were mixed at a mass ratio of 1:1, and the other steps and conditions were exactly the same as in Example 3 to obtain composite exosomes. Example 9

[0071] Directly encapsulate linear collagen.

[0072] Untreated linear type I collagen (85 kDa, purity ≥95%) was dissolved in water to prepare a 1 mg / mL solution (consistent with the collagen mass concentration of the nanoparticles in Example 1, showing bimodal or multimodal detection by DLS, with a particle size of 280-310 nm and a PDI of 0.5-0.8). This solution was directly mixed with exosomes derived from human umbilical cord mesenchymal stem cells at a mass ratio of 1:1 and processed under the same electroporation, purification, and lyophilization conditions as in Example 1 to obtain the sample. Example 10

[0073] Encapsulation efficiency determination.

[0074] The protein content in the supernatant before and after exosome encapsulation was determined using the BCA method, and the encapsulation efficiency (the ratio of protein content in the exosome precipitate to the total protein content) was calculated. The results are shown in Table 1. The encapsulation efficiency of the present invention (Examples 1-8) is significantly higher than that of the prior art (Example 9), and the encapsulation efficiency of Examples 1-6 is significantly higher than that of Examples 7-8, demonstrating the significant advantages of the present invention and its broad applicability to linear macromolecules of different molecular weights.

[0075] Table 1 Encapsulation efficiency Example Encapsulation rate % Example 1 85.6、39.2、87.3 Example 2 90.3、92.8、90.9 Example 3 88.7、89.2、91.8 Example 4 87.8、88.2、90.5 Example 5 86.5、89.3、87.4 Example 6 91.3、95.6、94.2 Example 7 40.5、38.2、41.3 Example 8 42.9、45.2、38.7 Example 9 4.2、5.1、3.9 Example 11

[0076] Particle size and dispersion uniformity were determined.

[0077] The particle size and PDI of the purified lyophilized exosomes reconstituted in physiological saline were determined by dynamic light scattering (DLS). PDI is a typical indicator of dispersion uniformity; generally, a PDI ≤ 0.2 indicates uniform dispersion. The results are shown in Table 2. The average particle size and PDI of the exosomes from this invention (Examples 1-8) are significantly smaller than those from the prior art (Example 9), and the average particle size and PDI of Examples 1-6 are significantly smaller than those of Examples 7-8, demonstrating the significant advantages of this method and its broad applicability to linear macromolecules of different molecular weights.

[0078] Table 2. Average particle size and polymeric dispersion (PDI) Example Average particle size (nm) PDI Peak characteristics Example 1 191±3.4 0.11±0.02 Single peak Example 2 192±3.9 0.10±0.01 Single peak Example 3 189±4.4 0.11±0.01 Single peak Example 4 184±4.1 0.10±0.01 Single peak Example 5 201±5.7 0.11±0.01 Single peak Example 6 190±2.1 0.10±0.02 Single peak Example 7 235±4.8 0.16±0.01 Single peak Example 8 242±7.3 0.16±0.02 Single peak Example 9 346±12.5 0.58±0.05 Bimodal or multimodal Example 12

[0079] Cell proliferation experiment.

[0080] Human skin fibroblasts (HDF) were co-cultured for 48 hours with PBS, blank exosomes, and exosomes prepared in Examples 1-9, respectively. Cell proliferation was detected by the CCK-8 assay. The results are shown in Table 3. The cell proliferation rate of the present invention (Examples 1-8) was significantly higher than that of the prior art (Example 9), and the cell proliferation rate of Examples 1-6 was significantly higher than that of Examples 7-8, demonstrating the significant functional synergistic effect of the present invention. The bioactivity of the composite exosomes of the present invention is not affected by the linear macromolecular molecular weight, exhibiting excellent adaptability. The composite exosomes of the present invention have a significant promoting effect on cell migration, providing key experimental evidence for wound healing and tissue repair.

[0081] Table 3 Cell proliferation rate Example Cell proliferation rate % Example 1 185.3±8.6 Example 2 192.5±10.4 Example 3 210.8±15.8 Example 4 178.9±9.3 Example 5 188.5±4.2 Example 6 203.4±11.7 Example 7 148.1±3.8 Example 8 152.3±4.7 Example 9 110.8±5.4 Blank exosomes 132.5±6.2 PBS The PBS group was 100 Example 13

[0082] Cell migration experiment.

[0083] HDF cells were seeded into 6-well plates. After the cell confluence reached 90%, a straight line (approximately 500 μm wide) was drawn along the bottom of the plate using a 200 μL pipette tip. The plates were washed with PBS to remove detached cells, and culture media containing PBS, blank exosomes, and exosomes from Examples 1-9 (final exosome / composite exosome concentration 0.2 mg / mL) were added. The plates were then incubated at 37°C in a 5% CO2 incubator. The scratched areas were photographed using an inverted microscope at 0 and 48 hours, and the scratch healing rate (healed area / initial scratch area × 100%) was calculated using ImageJ software. The results are shown in Table 4. The scratch healing rate of the present invention (Examples 1-8) was significantly higher than that of the prior art (Example 9), and the scratch healing rate of Examples 1-6 was significantly higher than that of Examples 7-8. This demonstrates the significant functional synergistic effect of the present invention. The bioactivity of the composite exosomes of the present invention is not affected by the linear macromolecular molecular weight, exhibiting excellent adaptability. The composite exosomes of the present invention significantly promote cell migration, providing crucial experimental evidence for wound healing and tissue repair.

[0084] Table 4 Scratch healing rate Example Scratch healing rate % Example 1 88.4±4.3 Example 2 80.1±5.1 Example 3 92.4±6.4 Example 4 91.2±3.4 Example 5 94.1±4.1 Example 6 93.5±6.3 Example 7 63.6±2.8 Example 8 64.8±4.6 Example 9 30.2±2.8 Blank exosomes 52.1±3.5 PBS 28.5±2.6 Example 14

[0085] Verification of exosome membrane integrity.

[0086] The exosomes prepared in Examples 1-9 were treated with 0.1% Triton. The particle number concentration before and after Triton treatment was detected using nanoflow cytometry, and the vesicle ratio was calculated as ((number of particles in the sample before Triton treatment - number of particles in the sample after Triton treatment) / number of particles in the sample before Triton treatment). A higher vesicle ratio indicates more exosomes with intact membranes. The results are shown in Table 5. The vesicle ratio of the present invention (Examples 1-8) is much higher than that of the prior art (Example 9), and the vesicle ratio of Examples 1-6 is significantly higher than that of Examples 7-8. This demonstrates the significant advantage of the present invention in effectively maintaining the integrity of the exosome membrane structure. The bioactivity of the composite exosomes of the present invention is not affected by the molecular weight of linear macromolecules, exhibiting excellent adaptability. The composite exosomes of the present invention have a significant promoting effect on cell migration, providing key experimental evidence for wound healing and tissue repair.

[0087] Table 5. Vesicle Ratio (Membrane Integrity) Example vesicle percentage Example 1 92.4±5.5 Example 2 91.2±6.1 Example 3 90.3±3.8 Example 4 89.1±5.4 Example 5 93.5±2.9 Example 6 89.4±4.8 Example 7 65.1±3.5 Example 8 61.3±2.9 Example 9 39.3±4.6 Example 15

[0088] Stability test.

[0089] The exosome lyophilized products of Examples 1-9 were stored at 2-8°C and samples were taken after 12 months to detect changes in particle size, PDI, and encapsulation efficiency, and to evaluate stability. The results are shown in Table 6. The stability of the technology of the present invention (Examples 1-8) is much higher than that of the prior art (Example 9), and the stability of Examples 1-6 is significantly higher than that of Examples 7-8. This demonstrates that the technology of the present invention has broad applicability to linear macromolecules of different molecular weights, meeting the actual needs of industrial production and product storage.

[0090] Table 6. Variations in average particle size, polymeric dispersion (PDI), and encapsulation efficiency. Example Average particle size increase rate % PDI Encapsulation retention rate % Example 1 <6 <0.15 95.3±3.1 Example 2 <6 <0.15 94.5±2.8 Example 3 <6 <0.15 96.7±3.5 Example 4 <6 <0.15 96.3±5.8 Example 5 <6 <0.15 97.1±6.2 Example 6 <6 <0.15 94.8±3.7 Example 7 12-16 0.15-0.2 62.1±3.7 Example 8 12-16 0.15-0.2 68.9±4.5 Example 9 25-30 >0.5 32.5±2.4 Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A composite exosome encapsulating linear macromolecular nanoparticles, characterized in that, The internal encapsulation consists of long-chain polymer nanoparticles with exosome membrane integrity ≥90%; the hydrodynamic diameter of the long-chain polymer nanoparticles is between 10 nm and 150 nm, the polymer dispersion is ≤0.15, and the surface is positively charged.

2. The composite exosome encapsulating linear macromolecular nanoparticles according to claim 1, characterized in that, The long-chain polymer is selected from one or more of polylactic acid and its derivatives, polycaprolactone, polydeoxyribonucleotides, polynucleotides, chitosan and its derivatives, silk fibroin, collagen, and hyaluronic acid.

3. The composite exosome encapsulating linear macromolecular nanoparticles according to claim 1, characterized in that, The long-chain polymer is collagen and / or hyaluronic acid, wherein the molecular weight of the collagen is 5 kDa-330 kDa and the molecular weight of the hyaluronic acid is 10 kDa-2200 kDa.

4. The method for preparing composite exosomes encapsulating linear macromolecular nanoparticles as described in any one of claims 1-3, characterized in that, Includes the following steps: 1) Prepare long-chain polymer nanoparticles using physical or chemical methods; 2) Mix nanoparticles and exosomes at a mass ratio of 1:10-10:1 in a buffer solution with pH 7.2-7.4 and incubate for 10-60 minutes at a mixing temperature of 4-25℃; 3) Nanoparticles are encapsulated inside exosomes using electroporation, freeze-thaw cycles, or ultrasound. 4) Purify the exosomes by ultracentrifugation or size exclusion chromatography to remove free nanoparticles and obtain composite exosomes.

5. The preparation method according to claim 4, characterized in that, In step 1): Physical methods include nanoprecipitation, electrostatic complexation, or thermally induced self-assembly. Nanoprecipitation uses microfluidic chips with a flow rate ratio of polymer solution to poor solvent of 1:0.5-1:

5. The chemical method is the cross-linking method; The long-chain polymers are collagen and / or hyaluronic acid. The nanoparticles have a hydrodynamic diameter of 10-150 nm, a PDI ≤ 0.15, and are positively charged by cationic amino acid modification. The molecular weight of collagen is 5 kDa-330 kDa, and the molecular weight of hyaluronic acid is 10 kDa-2200 kDa. Nanoparticles are positively charged by introducing cationic amino acids through blending or surface modification. The cationic amino acids are selected from one or more of lysine, arginine, and histidine.

6. The preparation method according to claim 4, characterized in that, In step 3): Electroporation method: voltage 300-600 V, capacitance 300-800 μF, pulse count 1-3 times, buffer conductivity ≤10 mS / cm; The freeze-thaw cycle method involves freezing in liquid nitrogen for 5-10 minutes, thawing in a 37°C water bath for 10-15 minutes, and repeating 1-5 times. The ultrasonic method uses a power of 50-150 W, operates for 3 seconds, pauses for 5 seconds, and has a total duration of 3-8 minutes. The temperature is controlled below 4℃.

7. The preparation method according to claim 5, characterized in that, When the molecular weight of collagen is ≤10 kDa and the molecular weight of hyaluronic acid is ≤50 kDa, nanoparticles are prepared by nanoprecipitation method, the polymer solution concentration is 0.5-5 mg / mL, and the flow rate ratio of polymer solution to poor solvent in microfluidic chip is 1:2-1:

5. When the molecular weight of collagen is ≥100 kDa and the molecular weight of hyaluronic acid is ≥500 kDa, nanoparticles are prepared by electrostatic complexation, with a cationic complexing agent volume ratio of 1:0.5-1:3 and an ultrasonication time of 5-30 minutes.

8. The use of a composite exosome encapsulating linear macromolecular nanoparticles as described in any one of claims 1-3 in the preparation of products for promoting tissue repair and regeneration.

9. The application according to claim 8, characterized in that, The product is a drug, medical device, cosmetic or food, and the tissue repair and regeneration is skin repair, anti-aging, bone and cartilage defect repair or wound healing.

10. The application according to claim 9, characterized in that, The product comprises a complex exosome as described in any one of claims 1-3, and a pharmaceutically acceptable carrier or a cosmetically acceptable matrix.

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