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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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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%&#8211;20% precast gel</td>
			<td>Genscript</td>
			<td>ExpressPlus PAGE GEL</td>
			<td>Western blotting</td>
		</tr>
		<tr>
			<td>5&#215; 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&#160;</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&#160;</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s modified Eagle&#8217;s medium</td>
			<td>Meilunbio</td>
			<td>MA0212</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;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&#160;</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>&#160;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&#160;</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&#160;</td>
		</tr>
		<tr>
			<td>Prism&#160;</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

1.3K 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 ...

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

We designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. While the device primarily functions as a bioreactor that mimics the native mechanical strains, it is also outfitted with a load cell for providing force feedback or mechanical testing of the constructs. The device subjects engineered cartilage constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and is compact enough to fit inside a standard tissue culture incubator. It loads samples directly in a tissue culture plate, and multiple plate sizes are compatible with the system. The device has been designed using components manufactured for precision-guided laser applications. Bi-axial loading is accomplished by two orthogonal stages. The stages have a 50 mm travel range and are driven independently by stepper motor actuators, controlled by a closed-loop stepper motor driver that features micro-stepping capabilities, enabling step sizes of less than 50 nm. A polysulfone loading platen is coupled to the bi-axial moving platform. Movements of the stages are controlled by Thor-labs Advanced Positioning Technology (APT) software. The stepper motor driver is used with the software to adjust load parameters of frequency and amplitude of both shear and compression independently and simultaneously. Positional feedback is provided by linear optical encoders that have a bidirectional repeatability of 0.1 &mu;m and a resolution of 20 nm, translating to a positional accuracy of less than 3 &mu;m over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities. In order to provide the force feedback to detect contact and evaluate loading responses, a precision miniature load cell is positioned between the loading platen and the moving platform. The load cell has high accuracies of 0.15% to 0.25% full scale.

We designed a novel mechanical loading bioreactor that can apply uniaxial or biaxial mechanical strain to a cartilage biocomposite prior to transplantation into an articular cartilage defect.

We  designed a loading device that is capable of applying uniaxial or biaxial  mechanical strain to a tissue engineered biocomposites fabricated for  ...

Microfluidic Fabrication of Polymeric and Biohybrid Fibers with Predesigned Size and Shape

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and used to dictate the shape as well as the diameter of a core stream.&#160;Grooves in the top and bottom of a microfluidic channel were designed to direct the sheath fluid and shape the core fluid.&#160;By matching the viscosity and hydrophilicity of the sheath and core fluids, the interfacial effects are minimized and complex fluid shapes can be formed.&#160;Controlling the relative flow rates of the sheath and core fluids determines the cross-sectional area of the core fluid.&#160;Fibers have been produced with sizes ranging from 300 nm to ~1 mm, and fiber cross-sections can be round, flat, square, or complex as in the case with double anchor fibers. Polymerization of the core fluid downstream from the shaping region solidifies the fibers.&#160;Photoinitiated click chemistries are well suited for rapid polymerization of the core fluid by irradiation with ultraviolet light.&#160;Fibers with a wide variety of shapes have been produced from a list of polymers including liquid crystals, poly(methylmethacrylate), thiol-ene and thiol-yne resins, polyethylene glycol, and hydrogel derivatives. Minimal shear during the shaping process and mild polymerization conditions also makes the fabrication process well suited for encapsulation of cells and other biological components.

Two adjacent fluids passing through a grooved microfluidic channel can be directed to form a sheath around a prepolymer core; thereby&#160;determining both shape and cross-section. Photoinitiated polymerization, such as thiol click chemistry, is well suited for rapidly solidifying the core fluid into a microfiber with predetermined size and shape.

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and ...

Porous Silicon Microparticles for Delivery of siRNA Therapeutics

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the successful implementation of siRNA therapy requires the use of delivery platforms that can overcome the major challenges of siRNA delivery, such as enzymatic degradation, low intracellular uptake and lysosomal entrapment. Here, a protocol for the preparation and use of a biocompatible and effective siRNA delivery system is presented. This platform consists of polyethylenimine (PEI) and arginine (Arg)-grafted porous silicon microparticles, which can be loaded with siRNA by performing a simple mixing step. The silicon particles are gradually degraded over time, thereby triggering the formation of Arg-PEI/siRNA nanoparticles. This delivery vehicle provides a means for protecting and internalizing siRNA, without causing cytotoxicity. The major steps of polycation functionalization, particle characterization, and siRNA loading are outlined in detail. In addition, the procedures for determining particle uptake, cytotoxicity, and transfection efficacy are also described.

Delivery remains the main challenge for the therapeutic implementation of small interfering RNA (siRNA). This protocol involves the use of a multifunctional and biocompatible siRNA delivery platform, consisting of arginine and polyethylenimine grafted porous silicon microparticles.

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the ...

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 ...

Fabrication and Characterization of a Conformal Skin-like Electronic System for Quantitative, Cutaneous Wound Management

Recent advances in the development of electronic technologies and biomedical devices offer opportunities for non-invasive, quantitative assessment of cutaneous wound healing on the skin. Existing methods, however, still rely on visual inspections through various microscopic tools and devices that normally include high-cost, sophisticated systems and require well trained personnel for operation and data analysis. Here, we describe methods and protocols to fabricate a conformal, skin-like electronics system that enables conformal lamination to the skin surface near the wound tissues, which provides recording of high fidelity electrical signals such as skin temperature and thermal conductivity. The methods of device fabrication provide details of step-by-step preparation of the microelectronic system that is completely enclosed with elastomeric silicone materials to offer electrical isolation. The experimental study presents multifunctional, biocompatible, waterproof, reusable, and flexible/stretchable characteristics of the device for clinical applications. Protocols of clinical testing provide an overview and sequential process of cleaning, testing setup, system operation, and data acquisition with the skin-like electronics, gently mounted on hypersensitive, cutaneous wound and contralateral tissues on patients.

This article presents methods to fabricate and characterize a conformal, skin-like electronic system and protocols for the use in clinical applications, particularly on cutaneous wound management.

Recent advances in the development of electronic technologies and biomedical devices offer opportunities for non-invasive, quantitative assessment of ...

Fabrication of Mechanically Tunable and Bioactive Metal Scaffolds for Biomedical Applications

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness and low bioactivity of metals. In this study, we have developed a new method towards fabricating a new type of bioactive and mechanically reliable porous metal scaffolds-densified porous Ti scaffolds. The method consists of two fabrication processes, 1) the fabrication of porous Ti scaffolds by dynamic freeze casting, and 2) coating and densification of the porous scaffolds. The dynamic freeze casting method to fabricate porous Ti scaffolds allowed the densification of porous scaffolds by minimizing the chemical contamination and structural defects. The densification process is distinctive for three reasons. First, the densification process is simple, because it requires a control of only one parameter (degree of densification). Second, it is effective, as it achieves mechanical enhancement and sustainable release of biomolecules from porous scaffolds. Third, it has broad applications, as it is also applicable to the fabrication of functionally graded porous scaffolds by spatially varied strain during densification.

Bioactive and mechanically reliable metal scaffolds have been fabricated through a method which consists of two processes, dynamic freeze casting for the fabrication of porous Ti, and coating and densification of the Ti scaffolds. The densification process is simple, effective and applicable to the fabrication of functionally graded scaffolds.

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness ...

Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus of this article is on the methods for fabricating ECM-derived porous foams and microcarriers for use as biologically-relevant substrates in advanced 3D in vitro cell culture models or as pro-regenerative scaffolds and cell delivery systems for tissue engineering and regenerative medicine. Using decellularized tissues or purified insoluble collagen as a starting material, the techniques can be applied to synthesize a broad array of tissue-specific bioscaffolds with customizable geometries. The approach involves mechanical processing and mild enzymatic digestion to yield an ECM suspension that is used to fabricate the three-dimensional foams or microcarriers through controlled freezing and lyophilization procedures. These pure ECM-derived scaffolds are highly porous, yet stable without the need for chemical crosslinking agents or other additives that may negatively impact cell function. The scaffold properties can be tuned to some extent by varying factors such as the ECM suspension concentration, mechanical processing methods, or synthesis conditions. In general, the scaffolds are robust and easy to handle, and can be processed as tissues for most standard biological assays, providing a versatile and user-friendly 3D cell culture platform that mimics the native ECM composition. Overall, these straightforward methods for fabricating customized ECM-derived foams and microcarriers may be of interest to both biologists and biomedical engineers as tissue-specific cell-instructive platforms for in vitro and in vivo applications.

The tissue-specific extracellular matrix (ECM) is a key mediator of cell function. This article describes methods for synthesizing pure ECM-derived foams and microcarriers that are stable in culture without the need for chemical crosslinking for applications in advanced 3D in vitro cell culture models or as pro-regenerative bioscaffolds.

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus ...

Fabrication and Characterization of Griffithsin-modified Fiber Scaffolds for Prevention of Sexually Transmitted Infections

Electrospun fibers (EFs) have been widely used in a variety of therapeutic applications; however, they have only recently been applied as a technology to prevent and treat sexually transmitted infections (STIs). Moreover, many EF technologies focus on encapsulating the active agent, relative to utilizing the surface to impart biofunctionality. Here we describe a method to fabricate and surface-modify poly(lactic-co-glycolic) acid (PLGA) electrospun fibers, with the potent antiviral lectin Griffithsin (GRFT). PLGA is an FDA-approved polymer that has been widely used in drug delivery due to its outstanding chemical and biocompatible properties. GRFT is a natural, potent, and safe lectin that possesses broad activity against numerous viruses including human immunodeficiency virus type 1 (HIV-1). When combined, GRFT-modified fibers have demonstrated potent inactivation of HIV-1 in vitro. This manuscript describes the methods to fabricate and characterize GRFT-modified EFs. First, PLGA is electrospun to create a fiber scaffold. Fibers are subsequently surface-modified with GRFT using 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS)chemistry. Scanning electron microscopy (SEM) was used to assess the size and morphology of surface-modified formulations. Additionally, a gp120 or hemagglutinin (HA)-based ELISA may be used to quantify the amount of GRFT conjugated to, as well as GRFT desorption from the fiber surface. This protocol can be more widely applied to fabricate fibers that are surface-modified with a variety of different proteins.

This manuscript describes the procedure to fabricate and characterize Griffithsin-modified poly(lactic-co-glycolic acid) electrospun fibers that demonstrate potent adhesive and antiviral activity against human immunodeficiency virus type 1 infection in vitro. Methods used to synthesize, surface-modify, and characterize the resulting morphology, conjugation, and desorption of Griffithsin from surface-modified fibers are described.

Electrospun fibers (EFs) have been widely used in a variety of therapeutic applications; however, they have only recently been applied as a technology to ...

Fabrication and Characterization of Optical Tissue Phantoms Containing Macrostructure

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By replicating the imaging environment, costly animal experiments to validate a technique may be circumvented. Predicting and optimizing the performance of in vivo and ex vivo imaging techniques requires testing on samples that are optically similar to tissues of interest. Tissue-mimicking optical phantoms provide a standard for evaluation, characterization, or calibration of an optical system. Homogenous polymer optical tissue phantoms are widely used to mimic the optical properties of a specific tissue type within a narrow spectral range. Layered tissues, such as the epidermis and dermis, can be mimicked by simply stacking these homogenous slab phantoms. However, many in vivo imaging techniques are applied to more spatially complex tissue where three dimensional structures, such as blood vessels, airways, or tissue defects, can affect the performance of the imaging system. 
This protocol describes the fabrication of a tissue-mimicking phantom that incorporates three-dimensional structural complexity using material with optical properties of tissue. Look-up tables provide India ink and titanium dioxide recipes for optical absorption and scattering targets. Methods to characterize and tune the material optical properties are described. The phantom fabrication detailed in this article has an internal branching mock airway void; however, the technique can be broadly applied to other tissue or organ structures.

Optical tissue phantoms are essential tools for calibration and characterization of optical imaging systems and validation of theoretical models. This article details a method for phantom fabrication that includes replication of tissue optical properties and three-dimensional tissue structure.

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By ...