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

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

1. Cell culture
   1. 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 Table of Materials) in Petri dishes with a diameter of 15 cm.
   2. Incubate ID8 cells in a CO₂ incubator (see Table of Materials) at 37 °C and 5% CO₂ until the cell density reaches 90%.
2. Isolation of exosomes
   1. Remove the culture medium from the Petri dishes and wash twice with Dulbecco's phosphate-buffered saline (DPBS). Add 20 mL of DMEM without fetal bovine serum (see Table of Materials) and penicillin-streptomycin solution (see Table of Materials). Continue the incubation at 37 °C and 5% CO₂ for 48 h.
   2. Collect the cell culture supernatant from 20 Petri dishes of 15 cm diameter. Centrifuge at 300 x g for 10 min at 4 °C and collect the supernatant for removing free cells.
   3. Centrifuge at 2000 x g for 10 min 4 °C, and collect the supernatant for removing cellular debris.
   4. Connect the 0.22 µm disposable vacuum filtration system (see Table of Materials) to the circulating water vacuum pump (see Table of Materials). Create a vacuum to allow the supernatant to pass through the 0.22 µm polyether sulfone membrane and enter the lower chamber of the filtration system, effectively removing vesicles or impurities larger than 0.22 µm from the supernatant.
      1. To connect the vacuum pump, turn on the pump switch and then connect it to the air inlet of the vacuum filtration system. When closing, disconnect the connection with the pump first and then turn off the vacuum pump. This procedure helps to prevent backflow of the vacuum pump and contamination of the culture fluid.
   5. Add 15 mL of supernatant to the inner tube of the 100 kDa ultrafiltration tube (see Table of Materials). Centrifuge at 3500 x g for 10 min at 4 °C, and remove the liquid from the outer tube.
      NOTE: Retain particles larger than 100 kDa on the inner tube membrane, while proteins smaller than 100 kDa will be centrifuged to the outer tube along with the solution. The exosomes were larger than 100 kDa and were aggregated on the inner tube membrane.
   6. Repeat the above steps until the total volume of the liquid is within 5 mL. Collect the liquid from the inner tube and add 1 mL of DPBS. Use a pipette to repeatedly pipette in the inner tube, ensuring the resuspension of the attached exosomes in DPBS, and then collect them.
   7. Centrifuge at 10000 x g for 60 min at 4 °C, and transfer the supernatant to a 6 mL quick-snap centrifuge tube (see Table of Materials). Seal the tube with a heat sealer.
   8. Centrifuge at 100,000 x g for 2 h 4 °C using an ultracentrifuge (see Table of Materials). Cut open the quick-snap centrifuge tube, remove the supernatant, and resuspend the pellet in 100 µL of DPBS. This is the exosome solution.
      NOTE: During the process of resuspending the pellet, it is necessary to pipette and agitate the tube wall at least 200 times using a pipette.

2. Fabrication of exo@MN

1. Preparation of master mold
   1. Construct a microneedle model with a 10 x 10 array using CAD software, featuring conical-shaped tips with the following parameters: a height of 1200 µm, a base diameter of 400 µm, and a pitch (distance between adjacent microneedles) of 900 µm.
   2. Use HTL resin as the material and print the master mold of the microneedle using a 3D printer (see Table of Materials).
   3. Immerse the master mold in absolute ethanol for 1 h to remove any resin adhering to the surface.
   4. Expose the mold to ultraviolet light for 5 min to cure it.
   5. Immerse the mold once again in absolute ethanol for 1 h, followed by drying it at 60 °C in a drying oven for 1 h. The master mold is ready.
2. Preparation of production mold
   1. Mix A and B components of polydimethylsiloxane (PDMS, see Table of Materials) in a ratio of 10:1. Stir well and pour the mixture into a master mold.
   2. Place the PDMS mold under a pressure of 1 psi for 15 min to remove air bubbles from the PDMS mixture.
   3. Cure the PDMS mold at 60 °C for 2 h and then demold to obtain the production mold of the microneedle.
   4. Clean the production mold with ultrapure water before use.
3. Preparation of solution
   1. Quantify the protein content of the exosome solution using a BCA assay kit (see Table of Materials). Add an appropriate amount of DPBS to achieve a concentration of 10 µg/µL for the exosome solution.
   2. Prepare a solution of 200 mg/mL trehalose (see Table of Materials) using DPBS as the solvent. Stir for 20 min to ensure full dissolution.
   3. Prepare a solution of 200 mg/mL hyaluronic acid (HA, see Table of Materials) using DPBS as the solvent. Stir overnight to ensure full dissolution.
   4. Prepare a solution of 150 mg/mL polyvinylpyrrolidone (PVP, see Table of Materials) using absolute ethanol as the solvent. Stir overnight to ensure full dissolution.
   5. Mix the exosome solution (10 µg/µL) and trehalose solution (200 mg/mL) in a 1:1 volume ratio. Then, add an equal volume of HA solution (200 mg/mL). Mix thoroughly to obtain the tip solution.
4. Fabrication of exo@MN
   1. Process the surface of the PDMS mold using a plasma cleaner (see Table of Materials) for 30 s at a low grade to enhance its hydrophilicity.
   2. Add 40 µL of the tip solution to the PDMS mold. Centrifuge at room temperature (RT) and 3000 x g for 3 min to ensure the liquid fills the needle layer of the mold.
   3. Remove excess liquid in the mold and dry at RT in a vacuum drying oven (see Table of Materials) for 1 day.
   4. Add 200 µL of PVP solution to the PDMS mold. Centrifuge 3000 x g for 3 min at RT to ensure the liquid fills the backing layer of the mold.
   5. Dry the mold at RT in a drying oven for 1 day, then demold to obtain the exo@MN patch.
      ​NOTE: Keep the exo@MN patch stored in a drying oven until ready for use.

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

1. Observation of morphology
   1. Paste the backing of the patch onto a 45° inclined surface and position it under a stereo microscope (see Table of Materials).
   2. Turn on the illuminator and capture the morphology of the patch using a 4x objective.
2. Scanning electron microscopy (SEM)
   1. Attach the patch to the sample stage using conductive adhesive and treat it with an auto fine coater (see Table of Materials) for 30 s.
   2. Capture images of the exo@MN patch using SEM (see Table of Materials) with an accelerating voltage of 1 kV.
3. Mechanical testing
   1. Cut out a 3 x 3 array from the patch and place it with the tips facing upwards on the rigid platform of a tensile meter (see Table of Materials). Adjust the height of the probe to bring it close to the needle tips without touching them.
   2. Set the parameters to stop when the pressure reaches 20 N automatically. Compress the needle tips vertically at a 0.5 mm/min speed and record the load versus displacement profile.
4. Dissolution
   1. Purchase fresh pig ears from the market and cut them into pieces (5 cm x 5 cm x 0.3 cm). Spread the pig skin on a table and use paper to dry the surface moisture.
   2. Prepare four dry microneedle patches with the needle tips facing down on the pig skin, spacing each patch slightly apart. Using the thumb and index fingers, vertically press down on each microneedle patch. Remove one patch every 15 s.
   3. Immediately place the removed patch in a drying oven and dry for 5 min. Cut off a column of microneedle tips, place them horizontally under a microscope (see Table of Materials), and use a 4x objective lens to observe the dissolution of the tips.

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.

1. Transmission electron microscopy (TEM)
   1. Treat the copper mesh (see Table of Materials) with a hydrophilic treatment using an ion cleaner (see Table of Materials) for 5 min.
   2. Dispense 10 µL of the sample onto the carbon side of the copper mesh. Let it stand for 1 min, then remove the liquid by blotting it with filter paper from the edges.
   3. Add 10 µL of 3% uranyl acetate (see Table of Materials) to the sample. Let it stand for 10 s, then remove the liquid by blotting with filter paper from the edges.
   4. Add 10 µL of 3% uranyl acetate to the sample. Let it stand for 1 min, then remove the liquid by blotting it with filter paper from the edges and air dry at RT.
   5. Capture images of the exosomes using TEM (see Table of Materials) with an accelerating voltage of 80 kV.
2. Nanoparticle tracking analysis (NTA)
   1. Dilute the exosome solution with DPBS to achieve a particle concentration of 1 x 10⁷ particles/mL. Inject slowly 1 mL of the exosome solution into the sample chamber of the NTA using 1 mL syringe (see Table of Materials).
   2. Set the instrument's standard operating procedure (SOP) type to EV-488 to detect the size distribution of the exosomes.
3. Western blotting (WB)
   1. Prepare the lysis buffer by mixing radioimmunoprecipitation assay (RIPA) lysis buffer: phenylmethanesulfonyl fluoride (see Table of Materials) in a ratio of 100:1. Lyse the exosomes on ice for 20 min, vortexing every 10 min.
   2. Centrifuge the solution at 12,000 x g for 10 min at 4 °C. Collect the supernatant to measure the protein concentration.
   3. Add 5x SDS-PAGE loading buffer (see Table of Materials) to the supernatant in a volume ratio of 1 : 4 and mix well. Heat at 100 °C for 10 min to denature the proteins.
   4. Add 5 µL of a 180 kDa pre-stained protein marker (see Table of Materials) and 10 µg of denatured sample to a 4%-20% precast gel (see Table of Materials). Run the electrophoresis system (see Table of Materials) at 60 V for 10 min and then at 140 V for 50 min.
   5. Transfer the proteins from the gel to a PVDF membrane (see Table of Materials) pre-activated with methanol. Perform the electrophoresis system at 290 mA for 90 min on ice.
   6. Incubate the PVDF membrane in 5% skim milk solution for 1 h. Wash three times with tris buffered saline with tween (TBST, see Table of Materials) for 5 min each.
   7. Cut out the target bands based on the position of the protein. Incubate the bands with anti-mouse CD63 antibody and anti-mouse Alix antibody (see Table of Materials) diluted to the appropriate concentration according to the manufacturer's instructions, and incubate overnight at 4 °C.
   8. Wash the bands three times with TBST. Incubate with HRP-conjugated anti-rabbit IgG (see Table of Materials) at RT for 1 h. Then, wash three times with TBST again.
   9. Apply a super ECL detection reagent (see Table of Materials) to cover the surface of the bands, and immediately expose and capture the bands using a gel imager (see Table of Materials).
4. Confocal laser scanning microscopy (CLSM)
   1. Cultivate 5 x 10⁵ DCs in a confocal dish using 2 mL of Roswell Park Memorial Institute 1640 (RPMI 1640) medium (see Table of Materials). Add exosomes or exo@MN, and incubate at 37 °C for 24 h.
   2. Add a drop of Hoechst 33342 to each confocal dish and incubate in the dark at 37 °C for 20 min.
   3. Perform imaging with a CLSM (see Table of Materials) using a 60x objective lens. Apply a drop of immersion oil (see Table of Materials) on the lens and place the confocal dish on the stage. Adjust the x/y/z axes to locate the appropriate focal plane of the cells.
   4. Initiate imaging with DAPI/TRITC/TD channels, select a resolution of 1024 px, and set the fast mode to 1/8 s.
5. Flow cytometry
   1. Cultivate DCs in a 6-well plate, each well containing 5 x 10⁵ cells and 2 mL of 1640 medium. Add equal volumes of solution of DPBS, exosomes, microneedles without exosomes, and exo@MN, respectively, and incubate at 37 °C for 24 h.
   2. Transfer the medium from each well into centrifugal tubes. Centrifuge at 300 x g for 3 min at 4 °C, and remove the supernatant.
   3. Add 0.5 µL of FITC-CD11c antibody, 2.5 µL of APC-CD80 antibody, and 0.5 µL of Pacific Blue I-A/I-E antibody (see Table of Materials) to 100 µL of 5% BSA solution (see Table of Materials). Mix well, resuspend the cells, and incubate on a shaker in ice and in the dark for 20 min.
   4. Add 1 mL of DPBS and centrifuge at 300 x g for 3 min at 4 °C, then remove the supernatant. Repeat the wash twice to remove unbound antibodies, and then proceed to analyze the corresponding fluorescent channels via flow cytometry (see Table of Materials).
      1. First, in the FSC-A/SSC-A scatter plot, gate the main cell population as P1. In the SSC-A/SSC-H scatter plot of the P1 cell population, gate a square P2 region along the diagonal to remove aggregated cells.
      2. Perform fluorescence analysis on the P2 cell population using the FITC, APC, and Pacific Blue channels. Set the stop condition to collect 10,000 cells in the P2 gate.

5. Statistical analysis

1. Express quantitative data as means ± standard deviations (SD). Assess statistical significance using t-test analysis using an appropriate data analysis software application. Consider p-values less than 0.05 to indicate statistical significance. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

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...

Materials

Name	Company	Catalog Number	Comments
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