Preparation method and application of glycosylation engineering exosome microneedle

Abstract

The invention discloses a preparation method and application of a glycosylation engineering exosome microneedle, and relates to the technical field of biological medicines. The preparation method comprises the following steps: modifying the membrane surface of an exosome with a phosphatidyl ethanolamine-polyethylene glycol-polysaccharide coupling compound to obtain a glycosylation engineering exosome; a solution of the glycosylation engineering exosome is used as a microneedle tip solution, a high polymer material solution is used as a microneedle substrate material solution, and the glycosylation engineering exosome microneedle is prepared by using a mold. The glycosylation engineering exosome microneedle prepared by the preparation method disclosed by the invention has good stability, and the exosome can still keep membrane integrity and biological activity in the microneedle preparation and storage process, so that safe, efficient, stable and continuous delivery of the exosome can be realized.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a method for preparing glycosylated engineered exosome microneedles and their applications. Background Technology

[0002] Exosomes derived from mammalian cells are an emerging class of biotherapeutic drugs and drug delivery vehicles, showing broad application prospects in drug delivery, disease diagnosis, tissue repair, and tumor immunotherapy. Microneedles, as a safe and efficient novel transdermal drug delivery technology, hold great potential for transdermal delivery of biopharmaceuticals. They can address the low bioavailability of orally administered drugs and the rapid clearance and unstable blood drug concentrations associated with injections, demonstrating significant clinical value in areas such as vaccination and insulin delivery.

[0003] Microneedle delivery of exosomes can effectively prolong the residence time of exosomes at the drug delivery site, thereby maintaining drug concentration and achieving sustained therapeutic effects. However, unlike traditional biologics, the unique vesicle structure and bioactivity of exosomes make them less stable, and they usually need to be stored at temperatures below -80°C. This leads to the risk of membrane rupture and inactivation of exosomes during microneedle preparation and delivery, which greatly hinders the industrialization prospects and clinical translation of exosome microneedles. To this end, the research team led by Professor Chang Hao at the Hangzhou Institute of Medical Sciences, Chinese Academy of Sciences, selected trehalose as the matrix material for the needle tip and loaded exosomes derived from mouse melanoma cells (B16-F10) to prepare an exosome microneedle vaccine. By comparing the differences in particle size, characteristic protein expression, and biological activity between exosomes in microneedles and fresh exosomes, they demonstrated that trehalose microneedles can effectively protect exosomes from enzymatic degradation and improve exosome stability (Mu, S., Qian, T., Li, Z., Zhao, P., Tao, Y., Chang, H., & Qu, F. (2024). Long-term storage and intradermal vaccination of tumor-derived exosomes via sugar microneedles for improving tumor immunotherapies. Chemical Engineering Journal, 497, 155595.). Meanwhile, cryo-microneedles prepared based on liquid nitrogen quick-freezing and freeze-drying processes (Xu, J., Lin, S., Chen, H., Yang, G., Zhou, M., Liu, Y., Li, A., Yin, S., & Jiang, X. (2024). Highly Active Frozen Nanovesicles Microneedles for Senile Wound Healing via Antibacteria, Immunotherapy, and Skin Regeneration. Adv Healthc Mater, 13(12), e2304315.) are another effective way to achieve stable loading of exosomes. Low temperature conditions are beneficial for the processing and storage of biopharmaceuticals. The above methods for improving the stability of exosome microneedles depend on specific matrix materials or processing techniques. How to form a "protective layer" through the engineering modification of exosomes themselves, so that they can withstand the conventional mold-based microneedle preparation process (room temperature environment, negative pressure conditions and drying stress), and be stably loaded into the microneedle system, is of great significance for expanding the application fields of exosome microneedles. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing glycosylated engineered exosome microneedles and their applications, thereby addressing the problems existing in the prior art. The glycosylated engineered exosome microneedles prepared using the method of this invention exhibit good stability, enabling exosomes to maintain membrane integrity and bioactivity during microneedle preparation and storage, thus achieving safe, efficient, stable, and continuous delivery of exosomes.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides a method for preparing glycosylated engineered exosome microneedles, comprising the following steps:

[0007] Phosphatidylethanolamine-polyethylene glycol-polysaccharide coupling compound was modified onto the membrane surface of exosomes to obtain glycosylated engineered exosomes.

[0008] The glycosylated engineered exosome microneedles were prepared using a mold with a solution of the glycosylated engineered exosomes as the microneedle tip solution and a polymer material solution as the microneedle base material solution.

[0009] Furthermore, the exosomes are mammalian cell-derived exosomes.

[0010] Furthermore, the polysaccharide in the phosphatidylethanolamine-polyethylene glycol-polysaccharide coupling compound is at least one of trehalose, dextran, chondroitin sulfate, chitosan, pullulan, hyaluronic acid, wolfberry polysaccharide, astragalus polysaccharide, bletilla polysaccharide, angelica polysaccharide, kelp polysaccharide, dendrobium officinale polysaccharide, ganoderma lucidum polysaccharide, and tremella polysaccharide.

[0011] Furthermore, the polymer material solution is at least one of polyvinyl alcohol solution, polyvinylpyrrolidone solution, hyaluronic acid solution, carboxymethyl cellulose solution, or hydroxypropyl methyl cellulose solution.

[0012] The present invention also provides a glycosylated engineered exosome microneedle prepared according to the above preparation method.

[0013] This invention also provides a method for preparing glycosylated drug-loaded exosome microneedles, comprising the following steps:

[0014] Drug-loaded exosomes are obtained by loading active drugs into exosomes through a drug loading reaction.

[0015] The membrane surface of the drug-loaded exosomes was modified with a phosphatidylethanolamine-polyethylene glycol-polysaccharide coupling compound to obtain glycosylated drug-loaded exosomes.

[0016] The glycosylated drug-loaded exosome microneedles were prepared using a mold with a solution of the glycosylated drug-loaded exosomes as the microneedle tip solution and a polymer material solution as the microneedle base material solution.

[0017] Furthermore, the drug loading reaction can be co-incubation, electroporation, ultrasound delivery, or iontophoresis.

[0018] Furthermore, the exosomes are mammalian cell-derived exosomes; and / or

[0019] The polysaccharide in the phosphatidylethanolamine-polyethylene glycol-polysaccharide coupling compound is at least one selected from trehalose, dextran, chondroitin sulfate, chitosan, pullulan, hyaluronic acid, wolfberry polysaccharide, astragalus polysaccharide, bletilla striata polysaccharide, angelica polysaccharide, kelp polysaccharide, dendrobium officinale polysaccharide, ganoderma lucidum polysaccharide, and tremella polysaccharide; and / or

[0020] The polymer solution is at least one of polyvinyl alcohol solution, polyvinylpyrrolidone solution, hyaluronic acid solution, carboxymethyl cellulose solution, or hydroxypropyl methyl cellulose solution.

[0021] The present invention also provides a glycosylated drug-loaded exosome microneedle prepared according to the above preparation method.

[0022] The present invention also provides the application of the above-mentioned glycosylated drug-loaded exosome microneedles in the preparation of microneedle drugs.

[0023] The present invention discloses the following technical effects:

[0024] The glycosylated engineered exosome microneedles provided by this invention aim to modify the exosome membrane with a phosphatidylethanolamine (DSPE)-polyethylene glycol (PEG)-polysaccharide coupling compound through lipid anchoring technology, forming a hydrophilic crown on the surface of nanoparticles, thereby significantly improving the stability of exosomes, ensuring their suitability for microneedle loading and preparation, and not affecting the normal biological activity of exosomes. Hydrophilic, flexible long-chain natural polysaccharides have a multi-hydroxyl structure. The polysaccharide chains grafted onto the surface of the exosome membrane extend outward to form a dense, highly hydrated hydrophilic "crown". This hydrophilic crown has the following functions: (1) It can increase the spatial distance, steric hindrance and steric repulsion between exosomes, and prevent the aggregation and precipitation of exosomes due to van der Waals forces, thereby preventing the fusion and aggregation of exosomes; (2) Exosomes are easily degraded by various proteases and nucleases in vivo and in vitro, and the dense structure of the polysaccharide crown acts as a physical barrier to hinder enzyme molecules. (2) Approaching and contacting bioactive molecules (such as proteins and RNA) on or inside the exosome membrane, thereby improving resistance to degradation by exogenous enzymes; (3) Hydrophilic surfaces can reduce non-specific interactions between hydrophobic or charged regions on the exosome membrane surface and the external environment (microneedle mold surface, container wall, etc.), thereby reducing adsorption and loss; (4) Natural polysaccharides have the ability to form a high-viscosity amorphous glassy state, which can help maintain the natural three-dimensional conformation of proteins during the drying and freezing of exosome solutions, thereby stabilizing the structure and activity of exosome membrane proteins during microneedle preparation.

[0025] This invention further utilizes glycosylated engineered exosomes to load active drugs, thereby enabling applications in the treatment of skin diseases and systemic diseases, vaccination, wound repair, and cancer diagnosis and treatment. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 A schematic diagram of the preparation process of glycosylated engineering exosome microneedles;

[0028] Figure 2 For DSPE-PEG 2000 -morphological images of HA-Exo microneedles; where a is a 3D digital microscope microneedle morphology image; b is an inverted fluorescence microscope microneedle morphology image;

[0029] Figure 3 For DSPE-PEG 2000-APS-Exo microneedle morphology and puncture performance images; a is a 3D digital microscope microneedle morphology image; b is an inverted fluorescence microscope microneedle morphology image; c is a result image of the skin puncture performance of microneedles using an ex vivo piglet skin model.

[0030] Figure 4 Transmission electron microscopy images of exosomes from Examples 1(a), 2(b), and Comparative Example 1(c);

[0031] Figure 5 The images show the Western Blot results of the microneedles prepared in Examples 1, 2 and Comparative Example 1.

[0032] Figure 6 The graph shows the exosome particle size detection results of the microneedles prepared in Examples 1, 2 and Comparative Example 1;

[0033] Figure 7 This is a transmission electron microscope image of exosomes from Example 6;

[0034] Figure 8 This is a transmission electron microscope image of exosomes from Example 7;

[0035] Figure 9 This is a transmission electron microscope image of exosomes from Example 8;

[0036] Figure 10 This is a transmission electron microscope image of exosomes from Comparative Example 2.

[0037] Figure 11 This is a transmission electron microscope image of exosomes from Comparative Example 3.

[0038] Figure 12 This is a transmission electron microscope image of exosomes from Comparative Example 4.

[0039] Figure 13 Laser confocal imaging of HA-Exo and Exo cells;

[0040] Figure 14 IC50 values ​​of B16-F10 cells after 24 h of treatment with different SAG (A), Exo / SAG (B), and HA-Exo / SAG (C) 50 The result of the value detection is shown in the figure. Detailed Implementation

[0041] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0042] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0043] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0044] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0045] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0046] Example 1: DSPE-PEG 2000 Preparation and characterization of hyaluronic acid-exosomal microneedles

[0047] This embodiment uses DSPE-PEG 2000 - Using hyaluronic acid (HA) as the grafting material, a highly stable glycosylated engineered exosome was prepared (see schematic diagram of the preparation process). Figure 1 The specific steps are as follows:

[0048] 1. Exosome extraction

[0049] Mouse melanoma B16-F10 cells were used. Cells were cultured in RPMI 1640 complete medium containing 10% fetal bovine serum (FBS) at 37°C and 5% CO2 under standard conditions. When cell confluence reached over 80%, the medium was replaced with serum-free RPMI 1640. After culturing for another 48 hours, the culture supernatant was collected. Exosomes (Exo) were extracted using ultracentrifugation, as follows:

[0050] Centrifuge at 4℃, 400×g for 5 min, and discard the precipitate;

[0051] Centrifuge at 2,000×g for 10 min at 4℃, and discard the precipitate;

[0052] Centrifuge at 4℃, 10,000×g for 30 min, and discard the precipitate;

[0053] Centrifuge at 100,000×g for 70 min at 4℃, discard the supernatant, and resuspend the precipitate in 10 mL PBS;

[0054] Centrifuge the resuspended solution again at 4℃ and 100,000×g for 70 min, discard the supernatant, and resuspend the precipitate in 400μL PBS to obtain the exosome suspension.

[0055] 2. Polysaccharide ligand grafting

[0056] The exosome suspension (0.8 mg / mL) obtained in step 1 was mixed with DSPE-PEG. 2000 -HA solution (1 mg / mL) at a mass ratio of Exo:DSPE-PEG 2000 Mix HA at a 1:1 ratio until homogeneous. Incubate at room temperature (25°C) for 24 hours. After incubation, centrifuge the mixture at 120,000 × g for 70 minutes at 4°C, discarding the supernatant to remove free DSPE-PEG. 2000 -HA. The precipitate was resuspended in pre-cooled PBS to obtain the HA-Exo solution (concentration 1.5 mg / mL).

[0057] 3. Layered microneedle preparation

[0058] Using the above-mentioned HA-Exo solution as the microneedle tip solution and a 20% (w / w) polyvinyl alcohol (PVA) solution (degree of polymerization 500-600) as the microneedle substrate material, microneedles were prepared. The preparation process is as follows:

[0059] A self-made vacuum plate was connected to a vacuum pump, and the negative pressure environment was set to -0.02 MPa. A polydimethylsiloxane (PDMS) mold was placed on the vacuum plate. 20 μL of HA-Exo solution was pipetted into each unit of the mold and spread evenly. Vacuum was maintained for 1 hour to ensure the solution fully filled the pinhole cavities under negative pressure. Then, 200 μL of 20% PVA solution was added as a base material to cover the needle tip layer. The microneedles were allowed to dry naturally at room temperature overnight to obtain the molded microneedles, named DSPE-PEG. 2000 -HA / Exo.

[0060] 4. Morphological characterization of microneedles

[0061] The morphology of microneedles was observed using a 3D stereomicroscope and an inverted fluorescence microscope. For example... Figure 2As shown, the microneedles prepared using glycosylated engineering exosome solution (HA-Exo) as the tip solution have good morphology after drying and demeasuring, exhibiting a smooth base layer, intact needle body, and full and sharp needle tip.

[0062] Example 2 DSPE-PEG 2000 Preparation and characterization of Astragalus polysaccharide-exosomal microneedles

[0063] This embodiment uses DSPE-PEG 2000 - Using Astragalus polysaccharide (APS) as the grafting material, a highly stable glycosylated engineered exosome was prepared. The specific steps are as follows:

[0064] 1. Exosome extraction

[0065] Exo was obtained by culturing mouse bone marrow mesenchymal stem cells (BMSCs) and then extracting the Exo suspension, using the same method as in Example 1.

[0066] 2. Polysaccharide ligand grafting

[0067] The Exo suspension (1 mg / mL) obtained in step 1 was mixed with DSPE-PEG. 2000 -APS solution (5 mg / mL) at a mass ratio of Exo:DSPE-PEG 2000 Mix APS at a ratio of 1:5 until homogeneous. Incubate at 37°C for 12 hours. After incubation, centrifuge the mixture at 4°C, 150,000 × g for 90 minutes, and discard the supernatant to remove free DSPE-PEG. 2000 -APS. The precipitate was resuspended in pre-cooled PBS to obtain the APS-Exo solution.

[0068] 3. Layered microneedle preparation

[0069] Using the aforementioned APS-Exo solution as the microneedle tip solution and a 40% (w / w) polyvinylpyrrolidone (PVP) solution as the microneedle substrate material, microneedles were prepared. The preparation process is as follows:

[0070] A self-made vacuum plate was connected to a vacuum pump, and the negative pressure environment was set to -0.08 MPa. A polydimethylsiloxane (PDMS) mold was placed on the vacuum plate. 40 μL of LAPS-Exo tip solution was drawn up using a pipette and injected into each unit of the mold, spreading it evenly. Vacuum was maintained for 2 hours to ensure the solution fully filled the needle cavity under negative pressure. Then, 200 μL of 40% PVP solution was added as a base material to cover the tip layer. The microneedles were then allowed to dry naturally at room temperature for 5 hours to obtain the molded microneedles, named DSPE-PEG. 2000 -APS / Exo.

[0071] 4. Morphology and skin puncture characteristics of microneedles

[0072] The morphology of the microneedles was observed using a 3D stereomicroscope and an inverted fluorescence microscope, and their skin puncture performance was tested. First, pigskin samples were removed from a -80℃ freezer and thawed at room temperature. After complete thawing, the pigskin was cut to a suitable size with a scalpel, and excess moisture was absorbed with filter paper. The pigskin was then unfolded with the stratum corneum facing upwards. The microneedles were carefully placed on the pigskin using tweezers, and a homemade needle inserter was used to press for 20 seconds. After removing the microneedles, the puncture area was stained with trypan blue solution for 30 minutes. After staining, excess staining agent was gently wiped away with a cotton swab. The treated skin samples were observed under an optical microscope, and the staining effect was recorded.

[0073] like Figure 3 As shown in Figures a and b, the microneedle array prepared using glycosylated engineered exosome solution (APS-Exo) as the tip solution is intact and has sharp tips. Figure 3 As shown in Figure c, the skin puncture performance of the microneedles was examined using an isolated piglet skin model. After removing the microneedles for 30 minutes, a complete array of pinholes was observed on the skin surface, indicating that the microneedles have good skin puncture performance.

[0074] Comparative Example 1: Preparation of non-glycosylated engineered exosome microneedles

[0075] Same as Example 1, except that the polysaccharide ligand grafting step is omitted, and Exo suspension is used directly as the microneedle tip solution.

[0076] Example 3: Morphological observation of exosome microneedles using transmission electron microscopy (TEM)

[0077] The morphology of Exo in the microneedles prepared in Examples 1, 2, and Comparative Example 1 was observed using transmission electron microscopy. The specific methods are as follows: The dried microneedles were removed from the mold and dissolved in an equal volume of PBS. After complete dissolution, 50 μL of Exo suspension was taken from each microneedle and fixed with 50 μL of 2% paraformaldehyde. 5 μL of the fixed sample was dropped onto a copper grid covered with a Formvar-carbon membrane and incubated at room temperature for 20 min. The copper grid (Formvar membrane side down) was washed twice with a drop of 100 μL PBS. The copper grid was then fixed with a drop of 50 μL of 1% glutaraldehyde for 5 min. Subsequently, the copper grid was washed eight times with 100 μL of deionized water, 2 min each time. The copper grid was negatively stained with a drop of 50 μL of 2% uranium acetate for 5 min. Finally, the copper grid was placed on ice and incubated with a drop of pre-cooled 50 μL methylcellulose for 10 min, then dried at room temperature for 10 min. The morphology was observed using TEM at an accelerating voltage of 80 kV.

[0078] like Figure 4 As shown, after reconstitution, Example 1 ( Figure 4 a) and Example 2 Figure 4In the glycosylated exosome microneedles of Example b), the exosomes maintained their morphology and exhibited typical cup-shaped or bowl-shaped vesicle structures. In contrast, Comparative Example 1 ( Figure 4 After the non-glycosylated microneedles in c) were reconstituted, the exosome membrane partially ruptured, and significant aggregation of exosomes was observed.

[0079] Example 4 verifies the protective effect of glycosylation engineering on exosomes in microneedles by measuring the expression of exosome marker proteins (Western Blot).

[0080] 1. The microneedles prepared in Examples 1, 2 and Comparative Example 1 were stored at 25°C for 7 days.

[0081] 2. Protein Sample Preparation: Microneedles from each group were reconstituted with an equal volume of PBS buffer. 500 μL of each microneedle solution was added to 200 μL of pre-chilled RIPA lysis buffer and 5 μL (1× final concentration) of a protease inhibitor mixture. Lysis was performed on ice for 30 min. The mixture was then centrifuged at 10,000×g, 4℃ for 10 min, and the supernatant was collected. An appropriate amount of supernatant was taken and 1 / 4 volume of 5×SDS-PAGE protein loading buffer was added. The mixture was mixed and boiled at 100℃ for 10 min. All samples were adjusted to the same protein concentration using 1×SDS-PAGE protein loading buffer.

[0082] 3. SDS-PAGE electrophoresis: Prepare a 10% separating gel and a 5% stacking gel.

[0083] 4. Sample loading: Pre-stained protein molecular weight standards and samples of each protein to be tested. Electrophoresis conditions: 80V constant voltage for 30 min, followed by 100V constant voltage for 90 min.

[0084] 5. Transfer membrane: Transfer membrane to PVDF membrane using a 100V constant voltage ice bath for 1 hour.

[0085] 6. Blocking: Block with rapid blocking solution for 15 min. Primary antibody incubation: After washing 3 times with TBST, add diluted primary antibody and incubate overnight at 4°C. Secondary antibody incubation: After washing 3 times with TBST, add diluted secondary antibody and incubate at room temperature for 1 hour, then wash 3 times with TBST.

[0086] 7. Chemiluminescence detection: ECL developer (solution A:solution B = 1:1) is dropped onto the PVDF membrane and developed using a chemiluminescence imaging system.

[0087] like Figure 5As shown, exosome microneedles prepared using different methods were stored at 25°C for 7 days, and then reconstituted to detect the expression levels of Exo marker proteins (TSG101, CD63, and Alix). The results showed that the expression levels of marker proteins TSG101, CD63, and Alix in glycosylated Exo (Examples 1 and 2) were significantly higher than those in unmodified Exo (Comparative Example 1). Notably, the expression level of Alix protein in the modified Exo group was not significantly different from that in freshly extracted Exo (P>0.05). This indicates that glycosylation modification can effectively maintain the structural integrity of exosomes, protect Exo proteins, and significantly improve the stability of Exo in microneedles.

[0088] Example 5 verifies the protective effect of glycosylation engineering on exosomes in microneedles (DLS) by measuring changes in exosome particle size.

[0089] The microneedles prepared in Examples 1, 2 and Comparative Example 1 were stored at 25°C for 7 days.

[0090] Each group of microneedles was reconstituted with an equal volume of PBS. 100 μL of the reconstituted microneedle solution was then diluted to 1 mL with PBS. The average particle size and particle size distribution were determined using a Malvern laser particle size analyzer (dynamic light scattering, DLS). Each sample was measured three times, and the average value was taken.

[0091] like Figure 6 As shown, after storing the microneedles at 25°C for 7 days, the particle size of Comparative Example 1 (Exo without glycosylation modification) changed significantly due to large particle aggregation and precipitation. However, the particle size distribution of Exo in the microneedles prepared after glycosylation modification (Examples 1 and 2) after reconstitution did not show significant changes compared to freshly extracted Exo. These results indicate that the mammalian cell exosome glycosylation modification method provided by this invention can effectively improve the stability of microneedles under storage conditions and effectively prevent Exo aggregation.

[0092] Example 6: Preparation of glycosylated engineered exosome microneedles derived from mouse hepatocellular carcinoma cells (Hepa1-6)

[0093] (1) Exosome extraction

[0094] Exo was extracted from mouse liver cancer cells (Hepa1-6) after culturing, and an Exo suspension was obtained, using the same method as in Example 1.

[0095] (2) Polysaccharide ligand grafting

[0096] Using DSPE-PEG 2000 - Lycium barbarum polysaccharide (LBP) was used as a grafting material for glycosylation engineering. The specific grafting concentration and conditions are detailed in Table 1.

[0097] (3) Layered microneedle preparation

[0098] Microneedles were prepared according to the tip matrix solution and substrate material specified in Table 1. The preparation process is as follows: A self-made vacuum pump was connected to a vacuum plate, and the negative pressure environment was set to -0.1 MPa. A polydimethylsiloxane (PDMS) mold was placed on the vacuum plate. 40 μL of tip solution was drawn up using a continuous pipette and injected into each unit of the mold, spreading it evenly. Vacuum was maintained for 4 hours to ensure that the solution fully filled the needle cavity under negative pressure. Subsequently, 100 μL of substrate material was added to cover the tip layer. The microneedles were then allowed to dry naturally at room temperature for about 4 hours to obtain the formed microneedles.

[0099] Example 7: Preparation of glycosylated engineered exosome microneedles derived from human malignant melanoma cells (A375)

[0100] (1) Exosome extraction

[0101] Exo was extracted from human malignant melanoma cells (A375) after culturing, and an Exo suspension was obtained, using the same method as in Example 1.

[0102] (2) Polysaccharide ligand grafting

[0103] Using DSPE-PEG 2000 - Trehalose (DTA) was used as a grafting material for glycosylation engineering. The specific grafting concentration and conditions are detailed in Table 1.

[0104] (3) Layered microneedle preparation

[0105] Microneedles were prepared according to the tip matrix solution and substrate material specified in Table 1. The preparation process is as follows: A self-made vacuum pump was connected to a vacuum plate, and the negative pressure environment was set to -0.1 MPa. A polydimethylsiloxane (PDMS) mold was placed on the vacuum plate. 40 μL of tip solution was drawn up using a continuous pipette and injected into each unit of the mold, spreading it evenly. Vacuum was maintained for 4 hours to ensure that the solution fully filled the needle cavity under negative pressure. Subsequently, 100 μL of substrate material was added to cover the tip layer. The microneedles were then allowed to dry naturally at room temperature for about 4 hours to obtain the formed microneedles.

[0106] Example 8: Preparation of glycosylated engineered exosome microneedles derived from human embryonic lung fibroblasts (HFL-1)

[0107] (1) Exosome extraction

[0108] Exo was extracted from human embryonic lung fibroblasts (HFL-1) after culturing, and an Exo suspension was obtained, using the same method as in Example 1.

[0109] (2) Polysaccharide ligand grafting

[0110] Using DSPE-PEG 2000- Chondroitin sulfate (CS) was used as a grafting material for glycosylation engineering. The specific grafting concentration and conditions are detailed in Table 1.

[0111] (3) Layered microneedle preparation

[0112] Microneedles were prepared according to the tip matrix solution and substrate material specified in Table 1. The preparation process is as follows: A self-made vacuum pump was connected to a vacuum plate, and the negative pressure environment was set to -0.1 MPa. A polydimethylsiloxane (PDMS) mold was placed on the vacuum plate. 40 μL of tip solution was drawn up using a continuous pipette and injected into each unit of the mold, spreading it evenly. Vacuum was maintained for 4 hours to ensure that the solution fully filled the needle cavity under negative pressure. Subsequently, 100 μL of substrate material was added to cover the tip layer. The microneedles were then allowed to dry naturally at room temperature for about 4 hours to obtain the formed microneedles.

[0113] Comparative Example 2

[0114] Same as Example 6, except that the polysaccharide ligand grafting step is omitted, and the polysaccharide solution and Exo suspension are mixed evenly according to the concentration ratio shown in Table 1 and then used directly as the needle tip solution.

[0115] Comparative Example 3

[0116] Same as Example 7, except that the polysaccharide ligand grafting step is omitted, and the polysaccharide solution and Exo suspension are mixed evenly according to the concentration ratio shown in Table 1 and then used directly as the needle tip solution.

[0117] Comparative Example 4

[0118] Same as Example 8, except that the polysaccharide ligand grafting step is omitted, and the polysaccharide solution and Exo suspension are mixed evenly according to the concentration ratio shown in Table 1 and then used directly as the needle tip solution.

[0119] Example 9

[0120] The microneedles prepared in Examples 6-8 and Comparative Examples 2-4 were stored at 30°C for 7 days. After that, the morphology of Exo in the microneedles was observed using a transmission electron microscope, and the specific operation was the same as in Example 3.

[0121] Table 1. Microneedle formulations and TEM results for Examples 6-8 and Comparative Examples 2-4

[0122]

[0123]

[0124] As shown in Table 1, the TEM results of the glycosylated Exo (Examples 6-8) were used to prepare microneedles. After storage at 30°C for 7 days, the microneedles were reconstituted and observed by TEM. Their morphology remained intact, displaying a clear lipid bilayer structure without aggregation or sedimentation. In contrast, the microneedles prepared by simply mixing the polysaccharide solution and exosome solution in Comparative Examples 2-4 showed Exo membrane rupture and significant aggregation after reconstitution. These results indicate that the glycosylated engineered exosome microneedles prepared in this invention can effectively improve the storage stability of exosomes, extend their shelf life, and maintain their biological activity, significantly broadening the application fields of exosomes.

[0125] Example 9: Preparation of Glycosylated Engineered Exosome Microneedles Loaded with Sanguisorbine via Co-incubation Method (Tumor Targeted Therapy)

[0126] 1. Exosome extraction

[0127] Exo was extracted from B16-F10 mouse melanoma cells to prepare an Exo suspension. The specific extraction steps were the same as step 1 in Example 1.

[0128] 2. Exosome drug delivery

[0129] The Exo suspension (2 mg / mL) obtained in step 1 was mixed with the drug component sanguinarine (SAG) (concentration 1 mg / mL) at a mass ratio of 1:1 and incubated at 37°C for 4 h. After incubation, the mixture was centrifuged at 120000×g at 4°C for 70 min, the supernatant was removed, and the centrifuged precipitate was resuspended in pre-cooled PBS to obtain the Exo / SAG solution (1.5 mg / mL).

[0130] 3. Preparation of drug-loaded exosomes by glycosylation engineering

[0131] The Exo / SAG solution (1.5 mg / mL) prepared in step 2 was mixed with DSPE-PEG. 2000 -HA solution (5 mg / mL) at a mass ratio of Exo / SAG:DSPE-PEG 2000 Mix the HA at a 1:1 ratio until homogeneous, and incubate at room temperature (25℃) for 24 hours. After incubation, centrifuge the mixture at 120,000×g for 70 minutes at 4℃, discarding the supernatant to remove free DSPE-PEG. 2000 -HA. The precipitate was resuspended in pre-cooled PBS to prepare an HA-Exo / SAG solution (concentration 2.5 mg / mL).

[0132] 4. Preparation of drug-loaded microneedles from exosomes via glycosylation engineering

[0133] Microneedles were prepared using HA-Exo / SAG solution as the microneedle tip solution and 40% PVA solution as the microneedle substrate material. The preparation process was as follows: A self-made vacuum pump was connected to a vacuum plate, and a negative pressure environment of -0.02 MPa was set. A polydimethylsiloxane (PDMS) mold was placed on the vacuum plate. 20 μL of HA-Exo / SAG tip solution was aspirated using a continuous pipette and injected into each unit of the mold, spreading it evenly. Vacuum was maintained for 1 hour to ensure that the solution fully filled the needle cavity under negative pressure. Subsequently, 200 μL of 40% PVA solution was added as the substrate material to cover the tip layer. The microneedles were then allowed to air dry overnight at room temperature to obtain the molded microneedles, named DSPE-PEG. 2000 -HA-Exo / SAG.

[0134] 5. Laser confocal microscopy was used to examine cellular uptake of HA-Exo.

[0135] B16-F10 cells were used at a rate of 1×10 5 B16-F10 cells were seeded at a density of 1 / mL in 35mm glass-bottom confocal culture dishes. After cell attachment, the supernatant was discarded, and the cells were washed twice with PBS. PKH26-labeled HA-Exo and Exo (50 μg / mL protein concentration) were added, and the cells were cultured for another 6 hours. The supernatant was aspirated, and the cells were fixed with 4% paraformaldehyde for 15 min, stained with DAPI in the dark for 10 min, washed three times with PBS, mounted with anti-fluorescence quenching mounting medium, and the uptake of HA-Exo and Exo by B16-F10 cells was observed under a laser confocal microscope. The mean intracellular fluorescence intensity was calculated using ImageJ.

[0136] 6. Cytotoxicity assay

[0137] B16-F10 cells were used at a rate of 1×10 4 The cells were seeded at a density of 1 / 2 well in a 96-well plate and incubated for 24 h. The supernatant was aspirated, and the cells were washed with PBS. 100 μL of different concentrations of Exo / SAG, SAG, and HA-Exo / SAG solutions were added to each well, with 6 replicates per group. The SAG concentrations in each group were 100, 200, 400, 600, and 800 ng / mL, respectively, and the cells were incubated for another 24 h. After 24 h, 10 μL of CCK-8 solution was added to each well, the plate was shaken for 30 s, and incubated for 2 h. The absorbance of each well at 450 nm was measured using a microplate reader. Cell viability was calculated as [(AS-Ab) / (Ac-Ab)] × 100%, and the cytotoxic effects of different formulations were investigated. As: experimental wells (containing cell culture medium, CCK-8, and toxic substance); Ab: blank wells (containing culture medium and CCK-8); Ac: control wells (containing cell culture medium, CCK-8, and no toxic substance). Based on the cell proliferation inhibition curve, the IC50 of different formulations was calculated using SPSS. 50 value.

[0138] Cellular uptake of exosomes is one of the main indicators for assessing their drug delivery function and therapeutic efficacy. For example... Figure 13 The laser confocal imaging results showed that HA-Exo exhibited a stronger green fluorescence signal on the cell membrane and in the cytoplasm of B16-F10 cells compared to the fluorescence intensity of Exo. These results demonstrate that B16-F10 cells have a stronger uptake effect of HA-Exo than Exo, possibly because B16-F10 cells with high CD44 expression have a stronger selectivity and affinity for HA-Exo, thereby enhancing cellular uptake.

[0139] like Figure 14 Cytotoxicity results showed that, compared with the control group, the SAG, Exo / SAG, and HA-Exo / SAG groups all exhibited concentration-dependent cytotoxicity and proliferation inhibition. IC50 50 The IC50 value measures the cytotoxicity of a drug; the higher the cytotoxicity, the lower the value. SAG, Exo / SAG, and HA-Exo / SAG have different IC50 values ​​for B16-F10 cells. 50 The concentrations were 631.8 ng / mL, 660.8 ng / mL, and 265.1 ng / mL, respectively, with IC50 values ​​of 631.8 ng / mL, 660.8 ng / mL, and 265.1 ng / mL. 50 The decreasing order indicates that its cytotoxicity increases sequentially. These results suggest that HA-Exo / SAG combines the homing effect of tumor-derived Exo with the advantages of both HA and SAG, exerting a targeted and synergistic tumor-killing effect.

[0140] This microneedle can be used for targeted therapy of melanoma (MM). Melanoma, a malignant tumor of the skin, is highly invasive and metastatic; it has a high incidence rate and poor prognosis, with a mortality rate of 70-90% in advanced stages, ranking first among malignant skin tumors. Current treatment methods for MM mainly include surgery, radiotherapy, chemotherapy, immunotherapy, and targeted therapy. Chemotherapy remains the primary treatment for melanoma, with main chemotherapy drugs including IFNα-2b, IL-2, dacarbazine, temozolomide, carboplatin, paclitaxel, and formustin. However, the efficacy of single-agent chemotherapy and combination chemotherapy is low, approximately 10-15%, and it damages the human immune system and has serious toxic side effects. Sanguisorba officinalis, as a monomeric component of traditional Chinese medicine, shows significant effects in inhibiting tumor cell proliferation, invasion, metastasis, and angiogenesis, and promoting tumor cell apoptosis, autophagy, and differentiation. Furthermore, because melanoma overexpresses CD44, and DSPE-PEG... 2000 -HA can serve as a targeting ligand for CD44. This receptor-binding ligand coupling engineering provides a cost-effective and scalable approach to improve tumor-specific homing while maintaining Exo stability. This allows for efficient delivery of loaded drugs to the tumor site to exert therapeutic effects.

[0141] Example 10: Preparation of Glycosylated Engineered Exosome Microneedles Loaded with Recombinant Human Collagen by Electroporation (Wound Repair Application)

[0142] 1. Exosome extraction

[0143] Human umbilical cord mesenchymal stem cells (hUC-MSCs) were cultured and Exo was extracted to prepare an Exo suspension. The specific extraction steps were the same as step 1 in Example 1, except that after ultrafiltration, the cells were resuspended in a low conductivity buffer solution (0.25M sucrose + 1mM EDTA (pH 7.4)).

[0144] 2. Exosome drug delivery

[0145] Recombinant human collagen (RHC) was diluted to 200 μg / mL with a low conductivity buffer solution (0.25 M sucrose + 1 mM EDTA (pH 7.4)) to obtain an RHC solution.

[0146] The Exo suspension (1.2 mg / mL) obtained in step 1 was mixed with the RHC solution (200 μg / mL) at a mass ratio of Exo:RHC = 1:1. Drug loading was then performed using electroporation. The mixture was operated on with the following parameters: 350 V, 100 μF, single pulse → incubation on ice for 10 min to restore membrane integrity. Finally, the mixture was centrifuged at 120,000 × g and 4 °C for 70 min to remove residual drug. The precipitate was resuspended in pre-cooled PBS to obtain the Exo / RHC drug-loaded solution.

[0147] 3. Preparation of drug-loaded exosomes by glycosylation engineering

[0148] The Exo / RHC drug loading solution (0.5 mg / mL) obtained in step 2 was mixed with DSPE-PEG. 2000 -APS solution (2.5 mg / mL) at a mass ratio of Exo / RHC:DSPE-PEG 2000 APS-Exo / RHC drug-loaded solution was prepared by mixing the APS and Exo components at a 1:1 ratio. The grafting steps were the same as step 3 in Example 9.

[0149] 4. Preparation of Glycosylated Drug-Loaded Exosome Microneedles

[0150] The microneedles were prepared using the above-mentioned APS-Exo / RHC drug loading solution as the microneedle tip solution and a 35% PVP solution as the microneedle substrate material. The specific preparation method is described in step 4 of Example 9.

[0151] This microneedle can be used for wound repair. Skin wounds are caused by various intrinsic pathological and external mechanical factors, leading to a series of local and even systemic physiological and pathological changes. Wound healing is a dynamic and highly continuous process, including hemostasis, inflammation, proliferation, and tissue remodeling. Exosomes loaded with RHC derived from hUC-MSCs can effectively promote wound repair. The core mechanism lies in the synergistic effect of exosomes as intelligent delivery carriers and RHCs as therapeutic commands, precisely regulating multiple key aspects of the wound healing process.

[0152] Example 11: Preparation of Glycosylated Exosome Microneedles Loaded with Dexamethasone (DSMX) Using Microfluidic Methods (Ocular Drug Delivery for the Treatment of Chronic Uveitis)

[0153] 1. Exosome extraction

[0154] Exo was extracted from hUC-MSCs cells after culturing, and an Exo suspension was prepared. The specific extraction steps were the same as step 1 in Example 1.

[0155] 2. Exosome drug delivery

[0156] A flow-focusing chip was used to premix the Exo suspension (2 mg / mL) obtained in step 1 with the dexamethasone drug solution (1.5 mg / mL). The Exo and dexamethasone were pre-incubated in a buffer solution containing 0.1% Triton X-100 for 10 min (temporarily increasing membrane permeability). Droplets were then generated, and the aqueous phase (premixed solution) was injected into the central channel (flow rate: 3 μL / min), and the oil phase (fluorinated oil (HFE 7500) + 2% surfactant (Pico-Surf™)) was injected into the sheath fluid channel (flow rate: 10 μL / min), generating droplets with a diameter of 80 μm. The droplets flowed through a 45°C temperature-controlled zone (2 cm in length, residence time: 1 min) to promote drug transmembrane transport. The droplets were collected, and a demulsifier (1H,1H,2H,2H-perfluorooctanol) was added. The aqueous phase was collected by centrifugation. Finally, residual oil phase and free drug were removed by ultrafiltration to obtain the Exo / DSMX solution.

[0157] 3. Preparation of drug-loaded exosomes by glycosylation engineering

[0158] The Exo / DSMX solution (1.2 mg / mL) obtained in step 2 was mixed with DSPE-PEG. 2000 -CS solution (4 mg / mL) at a mass ratio of Exo / DSMX:DSPE-PEG 2000 The CS-Exo / DSMX drug-loaded solution was prepared by mixing the CS and DSMX components at a 1:1 ratio. The grafting steps were the same as step 3 in Example 9.

[0159] 4. Preparation of Glycosylated Drug-Loaded Exosome Microneedles

[0160] Using the above-mentioned CS-Exo / DSMX drug-loaded solution as the microneedle tip solution and a 30% HA solution as the microneedle substrate material, microneedles were prepared. The specific preparation method is the same as step 4 of Example 9.

[0161] This microneedle can be used to treat chronic uveitis and for ocular drug delivery. Employing a chondroitin sulfate glycosylation grafting strategy, dexamethasone-loaded exosomes are precisely "locked" onto uveitis lesions after microneedle delivery, achieving long-lasting intelligent treatment.

[0162] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing glycosylated engineered exosome microneedles, characterized in that, Includes the following steps: Phosphatidylethanolamine-polyethylene glycol-polysaccharide coupling compound was modified onto the membrane surface of exosomes to obtain glycosylated engineered exosomes. The glycosylated engineered exosome microneedles were prepared using a mold with a solution of the glycosylated engineered exosomes as the microneedle tip solution and a polymer material solution as the microneedle base material solution.

2. The production method according to claim 1, characterized by, The exosomes are mammalian cell-derived exosomes.

3. The preparation method according to claim 1, characterized in that, The polysaccharide in the phosphatidylethanolamine-polyethylene glycol-polysaccharide coupling compound is at least one of trehalose, dextran, chondroitin sulfate, chitosan, pullulan, hyaluronic acid, wolfberry polysaccharide, astragalus polysaccharide, bletilla polysaccharide, angelica polysaccharide, kelp polysaccharide, dendrobium officinale polysaccharide, ganoderma lucidum polysaccharide, and tremella polysaccharide.

4. The production method according to claim 1, characterized by, The polymer solution is at least one of polyvinyl alcohol solution, polyvinylpyrrolidone solution, hyaluronic acid solution, carboxymethyl cellulose solution, or hydroxypropyl methyl cellulose solution.

5. A glycosylated engineered exosome microneedle prepared by the preparation method according to any one of claims 1-4.

6. A method of preparing glycosylated drug-loaded exosome microneedles, comprising: Includes the following steps: Drug-loaded exosomes are obtained by loading active drugs into exosomes through a drug loading reaction. The membrane surface of the drug-loaded exosomes was modified with a phosphatidylethanolamine-polyethylene glycol-polysaccharide coupling compound to obtain glycosylated drug-loaded exosomes. The glycosylated drug-loaded exosome microneedles were prepared using a mold with a solution of the glycosylated drug-loaded exosomes as the microneedle tip solution and a polymer material solution as the microneedle base material solution.

7. The preparation method according to claim 6, characterized in that, The drug loading reaction can be co-incubation, electroporation, ultrasound delivery, or iontophoresis.

8. The preparation method according to claim 6, characterized in that, The exosomes are mammalian cell-derived exosomes; and / or The polysaccharide in the phosphatidylethanolamine-polyethylene glycol-polysaccharide coupling compound is at least one selected from trehalose, dextran, chondroitin sulfate, chitosan, pullulan, hyaluronic acid, wolfberry polysaccharide, astragalus polysaccharide, bletilla striata polysaccharide, angelica polysaccharide, kelp polysaccharide, dendrobium officinale polysaccharide, ganoderma lucidum polysaccharide, and tremella polysaccharide; and / or The polymer solution is at least one of polyvinyl alcohol solution, polyvinylpyrrolidone solution, hyaluronic acid solution, carboxymethyl cellulose solution, or hydroxypropyl methyl cellulose solution.

9. A glycosylated drug-loaded exosome microneedle prepared by the preparation method according to any one of claims 6-8.

10. The use of the glycosylated drug-loaded exosome microneedles as described in claim 9 in the preparation of microneedle drugs.

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