F.L.Q. appreciates the support by supported by the Pioneer R&#38;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.

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
<ol>
	<li>Cell culture
	<ol>
		<li>Cultivate mouse ovarian epithelial cancer cells ID8 in Dulbecco&#39;s Modified Eagle Medium (DMEM) culture medium containing 10% fetal bovine serum and 1% penicillin-streptomycin solution (100&#215;) (see Table of Materials) in Petri dishes with a diameter of 15 cm.</li>
		<li>Incubate ID8 cells in a CO2 incubator (see Table of Materials) at 37 &#176;C and 5% CO2 until the cell density reaches 90%.</li>
	</ol>
	</li>
	<li>Isolation of exosomes
	<ol>
		<li>Remove the culture medium from the Petri dishes and wash twice with Dulbecco&#39;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 &#176;C and 5% CO2 for 48 h.</li>
		<li>Collect the cell culture supernatant from 20 Petri dishes of 15 cm diameter. Centrifuge at 300 x g for 10 min at 4 &#176;C and collect the supernatant for removing free cells.</li>
		<li>Centrifuge at 2000 x g for 10 min 4 &#176;C, and collect the supernatant for removing cellular debris.</li>
		<li>Connect the 0.22 &#181;m 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 &#181;m polyether sulfone membrane and enter the lower chamber of the filtration system, effectively removing vesicles or impurities larger than 0.22 &#181;m from the supernatant.
		<ol>
			<li>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.</li>
		</ol>
		</li>
		<li>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 &#176;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.</li>
		<li>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.</li>
		<li>Centrifuge at 10000 x g for 60 min at 4 &#176;C, and transfer the supernatant to a 6 mL quick-snap centrifuge tube (see Table of Materials). Seal the tube with a heat sealer.</li>
		<li>Centrifuge at 100,000 x g for 2 h 4 &#176;C using an ultracentrifuge (see Table of Materials). Cut open the quick-snap centrifuge tube, remove the supernatant, and resuspend the pellet in 100 &#181;L 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.</li>
	</ol>
	</li>
</ol>
2. Fabrication of exo@MN
<ol>
	<li>Preparation of master mold
	<ol>
		<li>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 &#181;m, a base diameter of 400 &#181;m, and a pitch (distance between adjacent microneedles) of 900 &#181;m.</li>
		<li>Use HTL resin as the material and print the master mold of the microneedle using a 3D printer (see Table of Materials).</li>
		<li>Immerse the master mold in absolute ethanol for 1 h to remove any resin adhering to the surface.</li>
		<li>Expose the mold to ultraviolet light for 5 min to cure it.</li>
		<li>Immerse the mold once again in absolute ethanol for 1 h, followed by drying it at 60 &#176;C in a drying oven for 1 h. The master mold is ready.</li>
	</ol>
	</li>
	<li>Preparation of production mold
	<ol>
		<li>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.</li>
		<li>Place the PDMS mold under a pressure of 1 psi for 15 min to remove air bubbles from the PDMS mixture.</li>
		<li>Cure the PDMS mold at 60 &#176;C for 2 h and then demold to obtain the production mold of the microneedle.</li>
		<li>Clean the production mold with ultrapure water before use.</li>
	</ol>
	</li>
	<li>Preparation of solution
	<ol>
		<li>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 &#181;g/&#181;L for the exosome solution.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Mix the exosome solution (10 &#181;g/&#181;L) 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.</li>
	</ol>
	</li>
	<li>Fabrication of exo@MN
	<ol>
		<li>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.</li>
		<li>Add 40 &#181;L 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.</li>
		<li>Remove excess liquid in the mold and dry at RT in a vacuum drying oven (see Table of Materials) for 1 day.</li>
		<li>Add 200 &#181;L 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.</li>
		<li>Dry the mold at RT in a drying oven for 1 day, then demold to obtain the exo@MN patch. 
		&#8203;NOTE: Keep the exo@MN patch stored in a drying oven until ready for use.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/67109/67109fig01.jpg" /> 
Figure 1: Fabrication process of exo@MN patches. <a href="https://www.jove.com/files/ftp_upload/67109/67109fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Characterization of exo@MN patches
<ol>
	<li>Observation of morphology
	<ol>
		<li>Paste the backing of the patch onto a 45&#176; inclined surface and position it under a stereo microscope (see Table of Materials).</li>
		<li>Turn on the illuminator and capture the morphology of the patch using a 4x objective.</li>
	</ol>
	</li>
	<li>Scanning electron microscopy (SEM)
	<ol>
		<li>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.</li>
		<li>Capture images of the exo@MN patch using SEM (see Table of Materials) with an accelerating voltage of 1 kV.</li>
	</ol>
	</li>
	<li>Mechanical testing
	<ol>
		<li>Cut out a 3 x 3&#160;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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Dissolution
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
4. Characterization of exosomes in exo@MN patch
NOTE: The microneedle tips of exo@MN are dissolved in 100 &#181;L of DPBS solution to perform the following characterization on the released exosomes.
<ol>
	<li>Transmission electron microscopy (TEM)
	<ol>
		<li>Treat the copper mesh (see Table of Materials) with a hydrophilic treatment using an ion cleaner (see Table of Materials) for 5 min.</li>
		<li>Dispense 10 &#181;L 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.</li>
		<li>Add 10 &#181;L 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.</li>
		<li>Add 10 &#181;L 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.</li>
		<li>Capture images of the exosomes using TEM (see Table of Materials) with an accelerating voltage of 80 kV.</li>
	</ol>
	</li>
	<li>Nanoparticle tracking analysis (NTA)
	<ol>
		<li>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).</li>
		<li>Set the instrument&#39;s standard operating procedure (SOP) type to EV-488 to detect the size distribution of the exosomes.</li>
	</ol>
	</li>
	<li>Western blotting (WB)
	<ol>
		<li>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.</li>
		<li>Centrifuge the solution at 12,000 x g for 10 min at 4 &#176;C. Collect the supernatant to measure the protein concentration.</li>
		<li>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 &#176;C for 10 min to denature the proteins.</li>
		<li>Add 5 &#181;L of a 180 kDa pre-stained protein marker (see Table of Materials) and 10 &#181;g 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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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&#39;s instructions, and incubate overnight at 4 &#176;C.</li>
		<li>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.</li>
		<li>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).</li>
	</ol>
	</li>
	<li>Confocal laser scanning microscopy (CLSM)
	<ol>
		<li>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 &#176;C for 24 h.</li>
		<li>Add a drop of Hoechst 33342 to each confocal dish and incubate in the dark at 37 &#176;C for 20 min.</li>
		<li>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.</li>
		<li>Initiate imaging with DAPI/TRITC/TD channels, select a resolution of 1024 px, and set the fast mode to 1/8 s.</li>
	</ol>
	</li>
	<li>Flow cytometry
	<ol>
		<li>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 &#176;C for 24 h.</li>
		<li>Transfer the medium from each well into centrifugal tubes. Centrifuge at 300 x g for 3 min at 4 &#176;C, and remove the supernatant.</li>
		<li>Add 0.5 &#181;L of FITC-CD11c antibody, 2.5 &#181;L of APC-CD80 antibody, and 0.5 &#181;L of Pacific Blue I-A/I-E antibody (see Table of Materials) to 100 &#181;L 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.</li>
		<li>Add 1 mL of DPBS and centrifuge at 300 x g for 3 min at 4 &#176;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).
		<ol>
			<li>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.</li>
			<li>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.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
5. Statistical analysis
<ol>
	<li>Express quantitative data as means &#177; 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 &#60; 0.05, **p &#60; 0.01, ***p &#60; 0.001, ****p &#60; 0.0001.</li>
</ol>

Microneedle Patches for Sustained Delivery of Exosomes in Biomedical Applications

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D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissolving+microneedles+for+long-term+storage+and+transdermal+delivery+of+extracellular+vesicles.">Dissolving microneedles for long-term storage and transdermal delivery of extracellular vesicles.</a> Biomaterials. 287, 121644 (2022).</li><li>Nir, Y., Potasman, I., Sabo, E., Paz, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fear+of+injections+in+young+adults:+Prevalence+and+associations.">Fear of injections in young adults: Prevalence and associations.</a> Am J Trop Med Hyg. 68 (3), 341-344 (2003).</li><li>Zhang, Y., Jiang, G., Yu, W., Liu, D., Xu, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microneedles+fabricated+from+alginate+and+maltose+for+transdermal+delivery+of+insulin+on+diabetic+rats.">Microneedles fabricated from alginate and maltose for transdermal delivery of insulin on diabetic rats.</a> Mater Sci Eng C Mater Biol Appl. 85, 18-26 (2018).</li><li>Roy, G., Garg, P., Venuganti, V. V. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microneedle+scleral+patch+for+minimally+invasive+delivery+of+triamcinolone+to+the+posterior+segment+of+eye.">Microneedle scleral patch for minimally invasive delivery of triamcinolone to the posterior segment of eye.</a> Int J Pharm. 612, 121305 (2022).</li><li>Creighton, R. L., Woodrow, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microneedle-mediated+vaccine+delivery+to+the+oral+mucosa.">Microneedle-mediated vaccine delivery to the oral mucosa.</a> Adv Healthc Mater. 8 (4), 1801180 (2019).</li><li>Hu, S., Zhu, D., Li, Z., Cheng, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detachable+microneedle+patches+deliver+mesenchymal+stromal+cell+factor-loaded+nanoparticles+for+cardiac+repair.">Detachable microneedle patches deliver mesenchymal stromal cell factor-loaded nanoparticles for cardiac repair.</a> ACS Nano. 16 (10), 15935-15945 (2022).</li><li>Yang, W., Zheng, H., Lv, W., Zhu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+status+and+prospect+of+immunotherapy+for+colorectal+cancer.">Current status and prospect of immunotherapy for colorectal cancer.</a> Int J Colorectal Dis. 38 (1), 266-276 (2023).</li><li>Zhang, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+and+technologies+for+exosome+isolation+and+characterization.">Methods and technologies for exosome isolation and characterization.</a> Small Methods. 2 (9), 1800021 (2018).</li><li>Bosch, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trehalose+prevents+aggregation+of+exosomes+and+cryodamage.">Trehalose prevents aggregation of exosomes and cryodamage.</a> Sci Rep. 6, 36162 (2016).</li><li>Bhattacharyya, M., Jariyal, H., Srivastava, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyaluronic+acid:+More+than+a+carrier,+having+an+overpowering+extracellular+and+intracellular+impact+on+cancer.">Hyaluronic acid: More than a carrier, having an overpowering extracellular and intracellular impact on cancer.</a> Carbohydr Polym. 317, 121081 (2023).</li><li>Chang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryomicroneedles+for+transdermal+cell+delivery.">Cryomicroneedles for transdermal cell delivery.</a> Nat Biomed Eng. 5 (9), 1008-1018 (2021).</li></ol>

H. C., F.L.Q, and S.J.M are inventors in a patent application that has been filed based on the data in this manuscript. H.C. is the scientific founder of Medcraft Biotech. Inc.

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...

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&#160;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&#160;(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&#160;(TEM), nanoparticle tracking analysis (NTA), and western blotting (WB). The internalization of exosomes by dendritic cells (DCs) was characterized using confocal laser scanning microscope&#160;(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.

<table><tbody><tr><td>100x penicillin-streptomycin solutions</td><td>Jrunbio Scientific</td><td>MA0110</td><td>Cell culture</td></tr><tr><td>180 kDa pre-stained protein marker</td><td>Thermo</td><td>26616</td><td>Western blotting</td></tr><tr><td>3% Uranyl acetate</td><td>Henan Ruixin Experimental Supplies</td><td>GZ02625</td><td>Morphological characterization of exosomes</td></tr><tr><td>3D printer</td><td>BMF technology</td><td>nanoArch S130</td><td>Mold preparation</td></tr><tr><td>4%&amp;ndash;20% precast gel</td><td>Genscript</td><td>ExpressPlus PAGE GEL</td><td>Western blotting</td></tr><tr><td>5&amp;times; SDS-PAGE loading buffer</td><td>Titan</td><td>04048254</td><td>Western blotting</td></tr><tr><td>Anti-mouse Alix antibody</td><td>Biolegend</td><td>12422-1-AP</td><td>Western blotting</td></tr><tr><td>Anti-mouse CD63 antibody</td><td>Biolegend</td><td>ab217345</td><td>Western blotting</td></tr><tr><td>APC anti-mouse CD80 antibody</td><td>Biolegend</td><td>104713</td><td>Antibody</td></tr><tr><td>Auto fine coater</td><td>ZIZHU</td><td>JBA5-100</td><td>Morphological characterization of microneedle</td></tr><tr><td>BCA assay kit</td><td>Beyotime</td><td>P0012</td><td>Protein concentration assay</td></tr><tr><td>Centrifuge</td><td>Thermo Fisher</td><td>Muitifuge X1R pro</td><td>Cell centrifuge</td></tr><tr><td>Circulating water vacuum pump</td><td>Yuhua Instrument</td><td>SHZ-D(III)</td><td>Filtration</td></tr><tr><td>CO2 incubator</td><td>Eppendorf</td><td>CellXpert C170</td><td>Cell culture</td></tr><tr><td>Confocal laser scanning microscope</td><td>Nikon</td><td>A1HD25</td><td>Fluorescence imaging</td></tr><tr><td>Copper mesh</td><td>Beijing Zhongjingkeyi Technology&amp;nbsp;</td><td>JF-ZJKY/300</td><td>Morphological characterization of exosomes</td></tr><tr><td>D- (+) -Trehalose dihydrate</td><td>Aladdin</td><td>5138-23-4</td><td>Fabrication of microneedle&amp;nbsp;</td></tr><tr><td>Dulbecco&amp;rsquo;s modified Eagle&amp;rsquo;s medium</td><td>Meilunbio</td><td>MA0212</td><td>Cell culture</td></tr><tr><td>Dulbecco&amp;rsquo;s phosphate-buffered saline</td><td>Meilunbio</td><td>MA0010</td><td>Cell culture</td></tr><tr><td>Electrophoresis system</td><td>Bio-rad</td><td>PowerPac-basic</td><td>Western blotting</td></tr><tr><td>Fetal bovine serum</td><td>Jrunbio Scientific</td><td>JR100</td><td>Cell culture</td></tr><tr><td>FITC anti-mouse CD11c antibody</td><td>Biolegend</td><td>117305</td><td>Antibody</td></tr><tr><td>Flow cytometry</td><td>BD</td><td>LSR Fortessa</td><td>Fluorescence detection</td></tr><tr><td>Gel imager</td><td>Cytiva</td><td>Amersham ImageQuant 800</td><td>Western blotting</td></tr><tr><td>HRP-conjugated anti-rabbit IgG</td><td>CST</td><td>7074S</td><td>Western blotting</td></tr><tr><td>HTL resin</td><td>BMF technology</td><td></td><td>Mold preparation</td></tr><tr><td>Hyaluronic acid (MW = 300 kDa)</td><td>Bloomage Biotechnology</td><td>9004-61-9</td><td>Fabrication of microneedle&amp;nbsp;</td></tr><tr><td>Immersion oil</td><td>Nikon</td><td>MXA22168</td><td>Fluorescence imaging</td></tr><tr><td>Ion cleaner</td><td>JEOL</td><td>EC-52000IC</td><td>Morphological characterization of exosomes</td></tr><tr><td>Microscope</td><td>Olympus</td><td>CKX53</td><td>Observe the microneedle tip dissolving process</td></tr><tr><td>Mouse ovarian epithelial cancer cell ID8</td><td>MeisenCTCC</td><td>&amp;nbsp;CC90105</td><td>Cell culture</td></tr><tr><td>Nanoparticle tracking analysis</td><td>Particle Metrix</td><td>ZetaView</td><td>Size analysis of exosomes</td></tr><tr><td>Pacific Blue anti-mouse I-A/I-E antibody</td><td>Biolegend</td><td>107619</td><td>Antibody</td></tr><tr><td>Phenylmethanesulfonyl fluoride</td><td>Beyotime</td><td>ST507</td><td>Protease inhibitors</td></tr><tr><td>Plasma cleaner</td><td>Hefei Kejing Material Technology</td><td>PDC-36G</td><td>Fabrication of microneedle&amp;nbsp;</td></tr><tr><td>Polydimethylsiloxane</td><td>Dow Corning</td><td>9016-00-6</td><td>Mold preparation</td></tr><tr><td>Polyvinylpyrrolidone (MW = 40 kDa)</td><td>Aladdin</td><td>9003-39-8</td><td>Fabrication of microneedle&amp;nbsp;</td></tr><tr><td>Prism&amp;nbsp;</td><td>GraphPad</td><td>Version 9</td><td>Statistical analysis</td></tr><tr><td>PVDF membrane</td><td>Millipore</td><td>IPVH00010</td><td>Western blotting</td></tr><tr><td>Quick-snap centrifuge</td><td>Beckman</td><td>344619</td><td>Exosomes extraction</td></tr><tr><td>RIPA lysis buffer</td><td>Applygen</td><td>C1053</td><td>Lysis membrane</td></tr><tr><td>Roswell park memorial institute 1640</td><td>Meilunbio</td><td>MA0548</td><td>Cell culture</td></tr><tr><td>Scanning electron microscope</td><td>JEOL</td><td>JSM-IT800</td><td>Morphological characterization of microneedle</td></tr><tr><td>Stereo microscope</td><td>Olympus</td><td>SZX16</td><td>Characterization of morphology</td></tr><tr><td>Super ECL detection reagent</td><td>Applygen</td><td>P1030</td><td>Western blotting</td></tr><tr><td>Tensile meter</td><td>Instron</td><td>68SC-05</td><td>Mechanical testing</td></tr><tr><td>Transmission electron microscope</td><td>JEOL</td><td>JEM-2100plus</td><td>Morphological characterization of exosomes</td></tr><tr><td>Tris buffered saline</td><td>Sangon Biotech</td><td>JF-A500027-0004</td><td>Western blotting</td></tr><tr><td>Tween-20</td><td>Beyotime</td><td>ST825</td><td>Western blotting</td></tr><tr><td>Ultracentrifuge</td><td>Beckman</td><td>Optima XPN-100</td><td>Exosomes extraction</td></tr><tr><td>Ultrafiltration tube</td><td>Millipore</td><td>UFC910096</td><td>Exosomes concentration</td></tr><tr><td>Vacuum drying oven</td><td>Shanghai Yiheng Technology</td><td>DZF-6024</td><td>Fabrication of microneedle</td></tr><tr><td>Vacuum filtration system</td><td>Biosharp</td><td>BS-500-XT</td><td>Filtration</td></tr></tbody></table>

fabrication and characterization of microneedle patches for loading and delivery of exosomes

Exosomes, as emerging &#34;next-generation&#34; 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.

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...

Author Spotlight: Innovative Microneedle-Based Strategies for Enhanced Exosome Delivery and Stability

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.

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

Exosomes, as emerging &#34;next-generation&#34; biotherapeutics and drug delivery vectors, hold immense potential in diverse biomedical fields, ranging ...

fabrication-characterization-microneedle-patches-for-loading-delivery

Research

JoVE Journal

Bioengineering

1.5K Views.  Tianjin University. 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.

Video: Author Spotlight: Innovative Microneedle-Based Strategies for Enhanced Exosome Delivery and Stability

Hollow Microneedle-based Sensor for Multiplexed Transdermal Electrochemical Sensing

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals suffering from chronic medical conditions facile assessment of their immediate physiological state. Furthermore, it could serve as a research tool for analysis of complex, multifactorial medical conditions. In order for such a multianalyte sensor to be realized, it must be minimally invasive, sampling of interstitial fluid must occur without pain or harm to the user, and analysis must be rapid as well as selective.
Initially developed for pain-free drug delivery, microneedles have been used to deliver vaccines and pharmacologic agents (e.g., insulin) through the skin.1-2 Since these devices access the interstitial space, microneedles that are integrated with microelectrodes can be used as transdermal electrochemical sensors. Selective detection of glucose, glutamate, lactate, hydrogen peroxide, and ascorbic acid has been demonstrated using integrated microneedle-electrode devices with carbon fibers, modified carbon pastes, and platinum-coated polymer microneedles serving as transducing elements.3-7,8
This microneedle sensor technology has enabled a novel and sophisticated analytical approach for in situ and simultaneous detection of multiple analytes. Multiplexing offers the possibility of monitoring complex microenvironments, which are otherwise difficult to characterize in a rapid and minimally invasive manner. For example, this technology could be utilized for simultaneous monitoring of extracellular levels of, glucose, lactate and pH,9 which are important metabolic indicators of disease states7,10-14 (e.g., cancer proliferation) and exercise-induced acidosis.15

This article details the construction of a multiplexed microneedle-based sensor. The device is being developed for in situ sampling and electrochemical analysis of multiple analytes in a rapid and selective manner. We envision clinical medicine and biomedical research uses for these microneedle-based sensors.

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals ...

Preparation of Exosomes for siRNA Delivery to Cancer Cells

Extracellular vesicles, in particular exosomes, have recently gained interest as novel drug delivery vectors due to their biological origin, abundance, and intrinsic capability in intercellular delivery of various biomolecules. This work establishes an isolation protocol to achieve high yield and high purity of exosomes for siRNA delivery. Human Embryonic Kidney cells (HEK-293 cells) are cultured in bioreactor flasks and the culture supernatant (hereon referred to as conditioned medium) is harvested on a weekly basis to allow for enrichment of HEK-293 exosomes. The conditioned medium (CM) is pre-cleared of dead cells and cellular debris by differential centrifugation and is subjected to ultracentrifugation onto a sucrose cushion followed by a washing step, to collect the exosomes. Isolated HEK-293 exosomes are characterized for yield, morphology and exosomal marker expression by nanoparticle tracking analysis, protein quantification, electron microscopy and flow cytometry, respectively. Small interfering RNA (siRNA), fluorescently labeled with Atto655, is loaded into exosomes by electroporation and excess siRNA is removed by gel filtration. Cell uptake in PANC-1 cancer cells, after 24 h incubation at 37 &#176;C, is confirmed by flow cytometry. HEK-293 exosomes are 107.0 &#177; 8.2 nm in diameter. The exosome yield and particle-to-protein ratio (P:P) ratio are 6.99 &#177; 0.22 &#215; 1012 particle/mL and 8.3 &#177; 1.7 &#215; 1010 particle/&#181;g, respectively. The encapsulation efficiency of siRNA in exosomes is ~ 10-20%. Forty percent of the cells show positive signals for Atto655 at 24 h post-incubation. In conclusion, exosome isolation by ultracentrifugation onto sucrose cushion offers a combination of good yield and purity. siRNA could be successfully loaded into exosomes by electroporation and subsequently delivered into cancer cells in vitro. This protocol offers a standard procedure for developing siRNA-loaded exosomes for efficient delivery to cancer cells.

An exosome is a new generation of drug delivery carriers. We established an exosome isolation protocol with high yield and purity for siRNA delivery. We also encapsulated fluorescently labelled non-specific siRNA into exosomes and investigated the cellular uptake of siRNA-loaded exosomes in cancer cells.

Extracellular vesicles, in particular exosomes, have recently gained interest as novel drug delivery vectors due to their biological origin, abundance, ...

Cancer Research

Combination of Microstereolithography and Electrospinning to Produce Membranes Equipped with Niches for Corneal Regeneration

Corneal problems affect millions of people worldwide reducing their quality of life significantly. Corneal disease can be caused by illnesses such as Aniridia or Steven Johnson Syndrome as well as by external factors such as chemical burns or radiation. Current treatments are (i) the use of corneal grafts and (ii) the use of stem cell expanded in the laboratory and delivered on carriers (e.g., amniotic membrane); these treatments are relatively successful but unfortunately they can fail after 3-5 years. There is a need to design and manufacture new corneal biomaterial devices able to mimic in detail the physiological environment where stem cells reside in the cornea. Limbal stem cells are located in the limbus (circular area between cornea and sclera) in specific niches known as the Palisades of Vogt. In this work we have developed a new platform technology which combines two cutting-edge manufacturing techniques (microstereolithography and electrospinning) for the fabrication of corneal membranes that mimic to a certain extent the limbus. Our membranes contain artificial micropockets which aim to provide cells with protection as the Palisades of Vogt do in the eye.

We report a technique for the fabrication of micropockets within electrospun membranes in which to study cell behavior. Specifically, we describe a combination of microstereolithography and electrospinning for the production of PLGA (Poly(lactide-co-glycolide)) corneal biomaterial devices equipped with microfeatures.

Corneal problems affect millions of people worldwide reducing their quality of life significantly. Corneal disease can be caused by illnesses such as ...

Polymeric Microneedle Array Fabrication by Photolithography

This manuscript describes the fabrication of polymeric microneedle (MN) arrays by photolithography. It involves a simple mold-free process by using a photomask consisting of embedded micro-lenses. Embedded micro-lenses were found to influence MN geometry (sharpness). Robust MN arrays with tip diameters ranging between 41.5 &#181;m &#177; 8.4 &#181;m and 71.6 &#181;m &#177; 13.7 &#181;m, with two different lengths (1,336 &#181;m &#177; 193 &#181;m and 957 &#181;m &#177; 171 &#181;m) were fabricated. These MN arrays may provide potential applications in delivery of low molecular and macromolecular therapeutic agents through skin.

Here, we present a protocol describing a mold-free fabrication process of the polymeric microneedles by photolithography.

This manuscript describes the fabrication of polymeric microneedle (MN) arrays by photolithography. It involves a simple mold-free process by using a ...

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

Manufacture and Drug Delivery Applications of Silk Nanoparticles

Silk is a promising biopolymer for biomedical and pharmaceutical applications due to its outstanding mechanical properties, biocompatibility and biodegradability, as well its ability to protect and subsequently release its payload in response to a trigger. While silk can be formulated into various material formats, silk nanoparticles are emerging as promising drug delivery systems. Therefore, this article covers the procedures for reverse engineering silk cocoons to yield a regenerated silk solution that can be used to generate stable silk nanoparticles. These nanoparticles are subsequently characterized, drug loaded and explored as a potential anticancer drug delivery system. Briefly, silk cocoons are reverse engineered first by degumming the cocoons, followed by silk dissolution and clean up, to yield an aqueous silk solution. Next, the regenerated silk solution is subjected to nanoprecipitation to yield silk nanoparticles &#8211; a simple but powerful method that generates uniform nanoparticles. The silk nanoparticles are characterized according to their size, zeta potential, morphology and stability in aqueous media, as well as their ability to entrap a chemotherapeutic payload and kill human breast cancer cells. Overall, the described methodology yields uniform silk nanoparticles that can be readily explored for a myriad of applications, including their use as a potential nanomedicine.

Nanoparticles are emerging as promising drug delivery systems for a broad range of indications. Here, we describe a simple yet powerful method to manufacture silk nanoparticles using reverse engineered Bombyx mori silk. These silk nanoparticles can be readily loaded with a therapeutic payload and subsequently explored for drug delivery applications.

Silk is a promising biopolymer for biomedical and pharmaceutical applications due to its outstanding mechanical properties, biocompatibility and ...

Fabrication of Fine Electrodes on the Tip of Hypodermic Needle Using Photoresist Spray Coating and Flexible Photomask for Biomedical Applications

We have introduced a fabrication method for electrical impedance spectroscopy (EIS)-on-a-needle (EoN: EIS-on-a-needle) to locate target tissues in the body by measuring and analyzing differences in the electrical impedance between dissimilar biotissues. This paper describes the fabrication method of fine interdigitated electrodes (IDEs) at the tip of a hypodermic needle using a photoresist spray coating and flexible film photomask in the photolithography process. A polyethylene terephthalate (PET) heat shrink tube (HST) with a wall thickness of 25 &#181;m is employed as the insulation and passivation layer. The PET HST shows a higher mechanical durability compared with poly(p-xylylene) polymers, which have been widely used as a dielectric coating material. Furthermore, the HST shows good chemical resistance to most acids and bases, which is advantageous for limiting chemical damage to the EoN. The use of the EoN is especially preferred for the characterization of chemicals/biomaterials or fabrication using acidic/basic chemicals. The fabricated gap and width of the IDEs are as small as 20 &#181;m, and the overall width and length of the IDEs are 400 &#181;m and 860 &#181;m, respectively. The fabrication margin from the tip (distance between the tip of hypodermic needle and starting point of the IDEs) of the hypodermic needle is as small as 680 &#181;m, which indicates that unnecessarily excessive invasion into biotissues can be avoided during the electrical impedance measurement. The EoN has a high potential for clinical use, such as for thyroid biopsies and anesthesia drug delivery in a spinal space. Further, even in surgery that involves the partial resection of tumors, the EoN can be employed to preserve as much normal tissue as possible by detecting the surgical margin (normal tissue that is removed with the surgical excision of a tumor) between the normal and lesion tissues.

The fabrication method for fine interdigitated electrodes (gap and width: 20 &#181;m) at the tip of a hypodermic needle (diameter: 720 &#181;m) is demonstrated using a spray coating and flexible film photomask in the photolithography process.

We have introduced a fabrication method for electrical impedance spectroscopy (EIS)-on-a-needle (EoN: EIS-on-a-needle) to locate target tissues in the ...

Neuroscience

Formulating and Characterizing an Exosome-based Dopamine Carrier System

Exosomes between 40 and 200 nm in size constitute the smallest subgroup of extracellular vesicles. These bioactive vesicles secreted by cells play an active role in intercellular cargo and communication. Exosomes are mostly found in body fluids such as plasma, cerebrospinal fluid, urine, saliva, amniotic fluid, colostrum, breast milk, joint fluid, semen, and pleural acid. Considering the size of exosomes, it is thought that they may play an important role in central nervous system diseases because they can pass through the blood-brain barrier (BBB). Hence, this study aimed to develop an exosome-based nanocarrier system by encapsulating dopamine into exosomes isolated from Wharton's jelly mesenchymal stem cells (WJ-MSCs). Exosomes that passed the characterization process were incubated with dopamine. The dopamine-loaded exosomes were recharacterized at the end of incubation. Dopamine-loaded exosomes were investigated in drug release and cytotoxicity assays. The results showed that dopamine could be successfully encapsulated within the exosomes and that the dopamine-loaded exosomes did not affect fibroblast viability.

Here, we aimed to obtain a formulation by dopamine loading of the isolated exosomes of stem cells from Wharton's jelly mesenchymal stem cells. Exosome isolation and characterization, drug loading into the resulting exosomes, and the cytotoxic activity of the developed formulation are described in this protocol.

Exosomes between 40 and 200 nm in size constitute the smallest subgroup of extracellular vesicles. These bioactive vesicles secreted by cells play an ...

Optimizing Extracellular Vesicle Delivery Using a Core-Sheath 3D-Bioprinted Scaffold for Chronic Wound Management

This study outlines a detailed protocol for the fabrication of core-sheath 3D-bioprinted scaffolds designed to enhance chronic wound healing. The protocol involves isolating extracellular vesicles (EVs) from mesenchymal stem cells (MSCs), known for their regenerative and immunomodulatory properties. These EVs are then incorporated into a unique scaffold structure. The scaffold features a core composed of alginate loaded with EVs, surrounded by a sheath made of carboxymethyl cellulose and alginate lyase. This innovative design ensures controlled scaffold degradation while promoting efficient and controlled release of EVs at the wound site. The protocol covers key steps, including the preparation and characterization of the EVs, the formulation of bio-inks for 3D bioprinting, and the optimization of printing parameters to achieve the desired core-sheath architecture. By combining structural integrity and bioactivity, the scaffold aims to address the limitations of conventional wound dressings, offering a targeted approach to accelerate tissue regeneration and reduce inflammation in chronic wounds. This method provides a reproducible and scalable strategy for developing advanced biomaterials with potential clinical applications in chronic wound management. The protocol also highlights critical considerations for achieving consistent results, ensuring adaptability for future therapeutic applications.

This study presents a protocol for fabricating core-sheath 3D-bio-printed scaffolds for chronic wound healing. Extracellular vesicles are isolated from mesenchymal stem cells, and loaded into the core (alginate) with the sheath made from carboxymethyl cellulose and alginate lyase. This design allows controlled scaffold degradation and efficient EVs release.

This study outlines a detailed protocol for the fabrication of core-sheath 3D-bioprinted scaffolds designed to enhance chronic wound healing. The protocol ...

Optimized Magnetic High-Yield Production of Endosome-Derived Nanovesicles

A Magnetic Separation-Assisted High-Speed Homogenization Method for Large-Scale Production of Endosome-Derived Vesicles

Extracellular vesicles (EVs) have attracted significant attention in physiological and pathological research, disease diagnosis, and treatment; however, their clinical translation has been limited by the lack of scale-up manufacturing approaches. Therefore, this protocol provides a magnetic separation-assisted high-speed homogenization method for the large-scale production of endosome-derived nanovesicles as a new type of exosome mimics (EMs) derived from the endosomes, which have about 100-time higher yield than conventional ultracentrifugation method. In this method, magnetic nanoparticles (MNPs) were internalized by parental cells via endocytosis and were subsequently accumulated within their endosomes. Then, MNPs-loaded endosomes were collected and purified by hypotonic treatment and magnetic separation. A high-speed homogenizer was utilized to break MNP-loaded endosomes into monodisperse nanovesicles. The resulting endosome-derived vesicles feature the same biological origin and structure, characterized by nanoparticle tracking analysis, transmission electron microscope, and western blotting. Their morphology and protein composition are similar to native EVs, indicating that EMs may potentially serve as a low-cost and high-yield surrogate of native EVs for clinical translations.

Author Spotlight: Development of a Large-Scale, Reproducible Production Method for Exosome Mimetics Using Magnetic Nanoparticles 

Here, we describe a magnetic separation-assisted high-speed homogenization method for large-scale production of endosome-derived nanovesicles as a new type of exosome mimics (EMs) that share the same biological origin and similar structure, morphology, and protein composition of native extracellular vesicles (EVs).

Extracellular vesicles (EVs) have attracted significant attention in physiological and pathological research, disease diagnosis, and treatment; however, ...