Exosomes possess significant clinical potential, but their practical application is limited due to easy in vivo clearance and poor stability. Microneedles present a solution by enabling localized delivery by puncturing physiological barriers and dry-state preservation, thereby addressing the limitations of exosome administration and expanding their clinical utility.
Exosomes, as emerging "next-generation" biotherapeutics and drug delivery vectors, hold immense potential in diverse biomedical fields, ranging from drug delivery and regenerative medicine to disease diagnosis and tumor immunotherapy. However, the rapid clearance by traditional bolus injection and poor stability of exosomes restrict their clinical application. Microneedles serve as a solution that prolongs the residence time of exosomes at the administration site, thereby maintaining the drug concentration and facilitating sustained therapeutic effects. In addition, microneedles also possess the ability to maintain the stability of bioactive substances. Therefore, we introduce a microneedle patch for loading and delivering exosomes and share the methods, including isolation of exosomes, fabrication, and characterization of exosome-loaded microneedle patches. The microneedle patches were fabricated using trehalose and hyaluronic acid as the tip materials and polyvinylpyrrolidone as the backing material through a two-step casting method. The microneedles demonstrated robust mechanical strength, with tips able to withstand 2 N. Pig skin was used to simulate human skin, and the tips of microneedles completely melted within 60 s after skin puncture. The exosomes released from the microneedles exhibited morphology, particle size, marker proteins, and biological functions comparable to those of fresh exosomes, enabling dendritic cells uptake and promoting their maturation.
Exosomes, which are small vesicles released by cells into the extracellular matrix, have been proposed as potential biotherapeutics and drug delivery vectors for the treatment of several diseases and cancers1. During their biogenesis process, exosomes encapsulate various biologically active molecules from within the cells, including functional proteins and nucleic acids2. As a result, when taken up by recipient cells during the transport process, exosomes have the ability to modulate gene expression and cellular functions in the target cells3. As a kind of natural information messenger, exosomes have been fully taken advantage of in tissue regeneration, immune regulation, and as a delivery carrier4. Through engineering techniques, specific ligands can be enriched on the surface of exosomes, enabling the induction or inhibition of signaling events in recipient cells or targeting specific cell types5. Chemotherapeutic agents can also be loaded into exosomes for cancer treatment6. Moreover, exosomes have the ability to cross the blood-brain barrier for therapeutic cargo delivery, making them highly promising for the treatment of brain disorders7. Compared to liposomes, exosomes exhibit enhanced cellular uptake and improved biocompatibility8. They are capable of efficiently entering other cells while demonstrating better tolerance and lower toxicity9. However, the traditional bolus injection of exosomes is prone to sequestration and rapid clearance by the liver, kidneys, and spleen in the bloodstream10. Moreover, exosomes have poor stability in vitro and are susceptible to storage conditions, which restrict their clinical applications11.
Microneedles, an array of micrometric-sized needle tips, have the capability to penetrate physiological barriers for the delivery of small molecule drugs12, proteins13, nucleic acids14, and nanomedicines15. Microneedles are precisely engineered to target lesions on the skin surface, and their dispersed tips ensure uniform drug distribution at the targeted site, thus amplifying their therapeutic impact16. The design and material composition of microneedles facilitate the dry storage of bioactive substances such as proteins and nucleic acids, enhancing their stability17. Traditional injection methods have a relatively short duration of action and can cause pain, inducing fear in patients18. The micrometer-sized length of microneedle minimizes tissue trauma and prevents nerve stimulation, thereby eliminating pain and improving patient compliance19. Additionally, the user-friendly nature of microneedles allows patients to self-administer the treatment without the need for specialized personnel16. In addition to the skin, microneedles can also be used in tissues such as the eyes20, oral mucosa21, heart22, and blood vessels23. The application of microneedles for the clinical delivery of exosomes provides a promising and prospective strategy.
Hence, we introduce an exosome-loaded microneedle (exo@MN) patch and disclose its fabrication method. The microneedle patches were fabricated using a two-step casting method, along with centrifugation and vacuum drying, which promotes the aggregation of exosomes at the microneedle tips, thereby enhancing delivery efficiency. Both the needle tips and backing were constructed using materials that exhibit excellent biocompatibility and water solubility. Trehalose and hyaluronic acid (HA) were incorporated as tip materials to provide protection for the exosomes, and polyvinylpyrrolidone (PVP) dissolved in absolute ethanol was chosen as the backing material. The morphology of the microneedle patch was characterized using microscopy and scanning electron microscope (SEM). The mechanical testing of the microneedle was assessed using a tensile meter to confirm their capability to penetrate the skin, and the release rate on pig skin was investigated to be 60 s. Furthermore, the morphology, size, and protein content of both fresh exosomes and exosomes in exo@MN were characterized using transmission electron microscope (TEM), nanoparticle tracking analysis (NTA), and western blotting (WB). The internalization of exosomes by dendritic cells (DCs) was characterized using confocal laser scanning microscope (CLSM), and the maturation of DCs was evaluated through flow cytometry. The morphological characterization and biological functions of the two types of exosomes are essentially consistent.
This study does not require ethical clearance as the pig skin used for the experiments described in section 3 was purchased as edible pig ears from the market and not sourced from experimental animals.
1. Isolation of exosomes
2. Fabrication of exo@MN
Figure 1: Fabrication process of exo@MN patches. Please click here to view a larger version of this figure.
3. Characterization of exo@MN patches
4. Characterization of exosomes in exo@MN patch
NOTE: The microneedle tips of exo@MN are dissolved in 100 µL of DPBS solution to perform the following characterization on the released exosomes.
5. Statistical analysis
Here, we present a protocol for the isolation of exosomes, fabrication and characterization of exo@MN patch. Figure 1 illustrates the process flowchart for the fabrication of exo@MN patch. The exosomes were mixed with trehalose and HA, and the mixture was then added to the microneedle mold and centrifuged. This process facilitated the aggregation of exosomes at the needle tips, promoting rapid release. After drying, PVP solution was added and centrifuged to fill the mold completely. Upon complete drying, the mold was removed to obtain the exo@MN patches. The morphology of the microneedles was characterized using microscopy and SEM, as shown in Figure 2. The tips in the exo@MN patch exhibited a conical shape arranged in a 10 x 10 array with sharp and intact needle tips. The tip of the microneedle can withstand a force of 2 N, allowing it to penetrate through the skin barrier. Furthermore, porcine skin was employed as a surrogate for human skin to simulate the application of exo@MN patches. The patches were pressed onto the pig skin to observe the release time, with the microneedle tips completely melting within 60 s. The exo@MN patches were dissolved in DPBS and characterized, as shown in Figure 3. TEM images captured vesicles with typical cup-shaped structures, consistent with the morphology of exosomes. The particle size distribution of the exo@MN solution by NTA revealed that the particles were predominantly concentrated within the range of 50-250 nm. However, there were also some larger particles present, which can be attributed to the adhesive properties of the HA material, causing exosome aggregation. WB analysis of the CD63/Alix proteins in the exo@MN solution demonstrated that exo@MN was capable of partially retaining proteins of exosomes. The biological functionality of exosomes released from the exo@MN was validated in Figure 4, focusing on the uptake of exosomes by cells and the activation of dendritic cells in comparison to fresh exosomes. CLSM images confirmed the efficient uptake of both fresh exosomes and exosomes in the exo@MN by DCs. Representative images and proportional statistics from flow cytometry demonstrate that the exosomes in exo@MN possess biological functions that promote the activation of DCs similar to fresh exosomes.
Figure 2: Characterization of exo@MN patches. (A) Stereo microscope images of exo@MN patches. Scale bar = 400 µm. (B) SEM image of exo@MN patches. Scale bar = 200 µm. (C) The load versus displacement profiles of exo@MN patch. (D) Microscopic images of exo@MN patches inserted into the pig skin at different times. Scale bar = 400 µm. Please click here to view a larger version of this figure.
Figure 3: Characterization of fresh exosomes and exosomes in exo@MN. (A) Morphology of exosomes by TEM. Scale bar = 100 nm. (B) Particle size distribution of exosomes by NTA. (C) CD63 and Alix of exosomes by WB. Please click here to view a larger version of this figure.
Figure 4: Biological testing of fresh exosomes and exosomes in exo@MN. (A) CLSM images of exosomes taken up by DCs. Scale bar = 10 µm. (B) Representative plots of activation of DCs by flow cytometry. (C) Quantification of percentage of CD11c+CD80+MHCII+ DCs. Data are presented as mean ± SD (n = 3). Please click here to view a larger version of this figure.
Currently, the main methods for isolating exosomes include ultracentrifugation, density-gradient centrifugation, ultrafiltration, precipitation, immunoaffinity magnetic beads, and microfluidics24. Due to the limited loading capacity of microneedles caused by their small needle tip space, it is necessary to increase the concentration of exosomes to load more. Therefore, we chose ultrafiltration to concentrate the cell culture supernatant and then used ultracentrifugation to isolate the exosomes. The concentration of exosomes was increased by a small amount of liquid resuspension so that the microneedles could be loaded with tens to hundreds of micrograms of exosomes. However, this method results in a significant presence of proteins in the isolated exosome solution. Additionally, a substantial loss of exosomes occurs during the centrifugation process. Nowadays, one of the limitations of the therapeutic application of exosomes is the lack of effective methods to isolate exosomes with high purity and in large quantities. As the clinical potential of exosomes is being increasingly explored, there is a need to develop more advanced techniques in the future to address the challenges associated with their extraction.
The key technology in this study is the design of exosome-based microneedles. In order to minimize the waste of exosomes and achieve stronger therapeutic effects, a two-step casting method was adopted to fabricate exo@MN patches. Trehalose has been previously reported as a suitable material for lyophilized preservation of exosomes25, while HA exhibits excellent biocompatibility, hydrophilicity, and functions in inflammation inhibition and skin repair promotion26. The fabrication of exosome-loaded needle tips using these two materials helps maintain the biological activity of exosomes, allowing for their rapid dissolution and release within the skin. Moreover, this approach minimizes the risk of infection and contributes to the swift recovery of the skin. Through centrifugation and vacuum drying, the exosomes were concentrated at the tips of the microneedles. Considering the incompatibility of ethanol and trehalose, PVP dissolved in absolute ethanol was selected as the backing material. This layered fabrication method effectively enhanced the delivery efficiency of the exosomes.
The poor stability of exosomes in vitro, whether stored at 4 °C, 37 °C, or -80 °C, may lead to changes in exosome structure or degradation11. In order to solve this problem, several studies have explored the storage methods of exosomes, such as freeze-drying. Microneedle technology provides another novel strategy. By drying at RT, microneedle technology can improve the stability of exosomes and can be directly used for exosome delivery without other treatments. Furthermore, the extraction process for exosomes is complex and prone to degradation, making large-scale applications challenging. Storing extracted exosomes using microneedle technology does not simplify the extraction steps but reduces the number of extractions required. A single extraction can produce a large quantity of exo@MN patches, which can be stored for an extended period. Utilizing exosome microneedles from the same batch for each treatment minimizes the potential impact of variations between different batches on therapeutic outcomes, thereby enhancing the repeatability and reliability of the exosome nanoplatform27. This provides more possibilities for the clinical application of exosomes.
In future research, it is worthwhile to explore methods that enable precise control of the payload and minimize drug variations between needle tips. This can be accomplished through process optimization, implementation of quality control measures, and utilization of advanced techniques. Addressing these aspects will significantly contribute to the development of dependable and consistent drug delivery systems.
The authors have nothing to disclose.
F.L.Q. appreciates the support by supported by the Pioneer R&D Program of Zhejiang (2022C03031), the National Key Research and Development Program of China (2021YFA0910103), the National Natural Science Foundation of China (22274141, 22074080), the Natural Science Foundation of Shandong Province (ZR2022ZD28) and the Taishan Scholar Program of Shandong Province (tsqn201909106). H.C. acknowledges the financial support from the National Natural Science Foundation of China (82202329). The authors acknowledge the use of instruments at the Shared Instrumentation Core Facility at the Hangzhou Institute of Medicine (HIM), Chinese Academy of Sciences.
100x penicillin-streptomycin solutions | Jrunbio Scientific | MA0110 | Cell culture |
180 kDa pre-stained protein marker | Thermo | 26616 | Western blotting |
3% Uranyl acetate | Henan Ruixin Experimental Supplies | GZ02625 | Morphological characterization of exosomes |
3D printer | BMF technology | nanoArch S130 | Mold preparation |
4%–20% precast gel | Genscript | ExpressPlus PAGE GEL | Western blotting |
5× SDS-PAGE loading buffer | Titan | 04048254 | Western blotting |
Anti-mouse Alix antibody | Biolegend | 12422-1-AP | Western blotting |
Anti-mouse CD63 antibody | Biolegend | ab217345 | Western blotting |
APC anti-mouse CD80 antibody | Biolegend | 104713 | Antibody |
Auto fine coater | ZIZHU | JBA5-100 | Morphological characterization of microneedle |
BCA assay kit | Beyotime | P0012 | Protein concentration assay |
Centrifuge | Thermo Fisher | Muitifuge X1R pro | Cell centrifuge |
Circulating water vacuum pump | Yuhua Instrument | SHZ-D(III) | Filtration |
CO2 incubator | Eppendorf | CellXpert C170 | Cell culture |
Confocal laser scanning microscope | Nikon | A1HD25 | Fluorescence imaging |
Copper mesh | Beijing Zhongjingkeyi Technology | JF-ZJKY/300 | Morphological characterization of exosomes |
D- (+) -Trehalose dihydrate | Aladdin | 5138-23-4 | Fabrication of microneedle |
Dulbecco’s modified Eagle’s medium | Meilunbio | MA0212 | Cell culture |
Dulbecco’s phosphate-buffered saline | Meilunbio | MA0010 | Cell culture |
Electrophoresis system | Bio-rad | PowerPac-basic | Western blotting |
Fetal bovine serum | Jrunbio Scientific | JR100 | Cell culture |
FITC anti-mouse CD11c antibody | Biolegend | 117305 | Antibody |
Flow cytometry | BD | LSR Fortessa | Fluorescence detection |
Gel imager | Cytiva | Amersham ImageQuant 800 | Western blotting |
HRP-conjugated anti-rabbit IgG | CST | 7074S | Western blotting |
HTL resin | BMF technology | Mold preparation | |
Hyaluronic acid (MW = 300 kDa) | Bloomage Biotechnology | 9004-61-9 | Fabrication of microneedle |
Immersion oil | Nikon | MXA22168 | Fluorescence imaging |
Ion cleaner | JEOL | EC-52000IC | Morphological characterization of exosomes |
Microscope | Olympus | CKX53 | Observe the microneedle tip dissolving process |
Mouse ovarian epithelial cancer cell ID8 | MeisenCTCC | CC90105 | Cell culture |
Nanoparticle tracking analysis | Particle Metrix | ZetaView | Size analysis of exosomes |
Pacific Blue anti-mouse I-A/I-E antibody | Biolegend | 107619 | Antibody |
Phenylmethanesulfonyl fluoride | Beyotime | ST507 | Protease inhibitors |
Plasma cleaner | Hefei Kejing Material Technology | PDC-36G | Fabrication of microneedle |
Polydimethylsiloxane | Dow Corning | 9016-00-6 | Mold preparation |
Polyvinylpyrrolidone (MW = 40 kDa) | Aladdin | 9003-39-8 | Fabrication of microneedle |
Prism | GraphPad | Version 9 | Statistical analysis |
PVDF membrane | Millipore | IPVH00010 | Western blotting |
Quick-snap centrifuge | Beckman | 344619 | Exosomes extraction |
RIPA lysis buffer | Applygen | C1053 | Lysis membrane |
Roswell park memorial institute 1640 | Meilunbio | MA0548 | Cell culture |
Scanning electron microscope | JEOL | JSM-IT800 | Morphological characterization of microneedle |
Stereo microscope | Olympus | SZX16 | Characterization of morphology |
Super ECL detection reagent | Applygen | P1030 | Western blotting |
Tensile meter | Instron | 68SC-05 | Mechanical testing |
Transmission electron microscope | JEOL | JEM-2100plus | Morphological characterization of exosomes |
Tris buffered saline | Sangon Biotech | JF-A500027-0004 | Western blotting |
Tween-20 | Beyotime | ST825 | Western blotting |
Ultracentrifuge | Beckman | Optima XPN-100 | Exosomes extraction |
Ultrafiltration tube | Millipore | UFC910096 | Exosomes concentration |
Vacuum drying oven | Shanghai Yiheng Technology | DZF-6024 | Fabrication of microneedle |
Vacuum filtration system | Biosharp | BS-500-XT | Filtration |