Summary

Fabrication and Characterization of Microneedle Patches for Loading and Delivery of Exosomes

Published: July 12, 2024
doi:

Summary

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.

Abstract

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.

Introduction

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.

Protocol

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 Cell culture Cultivate mouse ovarian epithelial cancer cells ID8 in Dulbecco's Modified Eagle Medium (DMEM) culture medium containing 10% fetal bovine serum and 1% penicillin-streptomycin solution (100×) (see Tabl…

Representative Results

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 com…

Discussion

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. Th…

Disclosures

The authors have nothing to disclose.

Acknowledgements

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.

Materials

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

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Cite This Article
Mu, S., Chang, H., Qu, F. Fabrication and Characterization of Microneedle Patches for Loading and Delivery of Exosomes. J. Vis. Exp. (209), e67109, doi:10.3791/67109 (2024).

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