Name: 作者聚焦：用于增强外泌体递送和稳定性的创新微针策略
Uploaded: 2024-07-12T14:55:00
Description: Exosomes, as emerging &#34;next-generation&#34; biotherapeutics and drug delivery vectors, hold immense potential in diverse biomedical fields, ranging ...

F.L.Q. 感谢浙江省先锋研发计划（2022C03031）、国家重点研发计划（2021YFA0910103）、国家自然科学基金（22274141,22074080）、山东省自然科学基金（ZR2022ZD28）和山东省泰山学者计划（tsqn201909106）的支持。H.C. 感谢中国国家自然科学基金 （82202329） 的财政支持。作者承认了中国科学院杭州医学研究所 （HIM） 共享仪器核心设施的仪器使用情况。

这项研究不需要道德审查，因为用于第 3 节中描述的实验的猪皮是从市场上作为可食用的猪耳朵购买的，而不是来自实验动物。
1. 外泌体的分离
<ol><li>细胞培养<ol><li>在直径为 15 cm 的培养皿中，在含有 10% 胎牛血清和 1% 青霉素 - 链霉素溶液 （100×） 的 Dulbecco 改良 Eagle 培养基 （DMEM） 培养基中培养小鼠卵巢上皮癌细胞 ID8（参见 材料表）。</li>
		<li>...

用于生物医学应用中外泌体持续递送的微针贴片

<ol>
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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>

目前，分离外泌体的主要方法包括超速离心、密度梯度离心、超滤、沉淀、免疫亲和磁珠和微流控24。由于微针尖端空间小导致负载能力有限，因此需要增加外泌体的浓度以加载更多。因此，我们选择超滤浓缩细胞培养上清液，然后用超速离心分离外泌体。通过少量液体重悬来增加外泌体的浓度，以便微针可以加载数十至数百微克的外泌体。然而，这种方法导致分离的外泌体溶?...

外泌体是细胞释放到细胞外基质中的小囊泡，已被提议作为治疗多种疾病和癌症的潜在生物治疗药物和药物递送载体1。在其生物发生过程中，外泌体封装了细胞内的各种生物活性分子，包括功能性蛋白和核酸2。因此，当外泌体在运输过程中被受体细胞吸收时，外泌体具有调节靶细胞中基因表达和细胞功能的能力3。作为一种天然信息信使，外泌体在组织再生、免疫调节和作为递送载体中得到了充分利用4。通过工程技术，可以在外泌体表面富集特异性配体，从而能够诱导或抑制受体细胞中的信号转导事件或靶向特定细胞类型5。化疗剂也可以加载到外泌体中用于癌症治疗6。此外，外泌体能够穿过血脑屏障进行治疗性货物递送，使其在治疗脑部疾病方面非常有前途7。与脂质体相比，外泌体表现出增强的细胞摄取和更好的生物相容性8。它们能够有效地进入其他细胞，同时表现出更好的耐受性和更低的毒性9。然而，传统的外泌体推注容易被血流中的肝脏、肾脏和脾脏隔离和快速清除10。此外，外泌体 在体外 稳定性差，易受储存条件的影响，这限制了它们的临床应用11。
微针是一系列微米大小的针尖，能够穿透生理屏障，以递送小分子药物12、蛋白质13、核酸14 和纳米药物15。微针经过精确设计，可针对皮肤表面的病变，其分散的尖端可确保药物在目标部位均匀分布，从而放大其治疗效果16。微针的设计和材料组成有助于蛋白质和核酸等生物活性物质的干储存，从而提高其稳定性17。传统的注射方法作用持续时间相对较短，会引起疼痛，使患者感到恐惧18。微米级的微针长度可最大限度地减少组织创伤并防止神经刺激，从而消除疼痛并提高患者的依从性19。此外，微针的用户友好性使患者无需专业人员即可自行进行治疗16。除了皮肤，微针还可用于眼睛20、口腔粘膜21、心脏22 和血管23 等组织。微针在外泌体临床递送中的应用提供了一种有前途且具有前瞻性的策略。
因此，我们介绍了一种加载外泌体的微针 （exo@MN） 贴片并公开了其制造方法。微针贴片采用两步铸造法制备，同时离心和真空干燥，促进外泌体在微针尖端聚集，从而提高递送效率。针尖和背衬均使用具有优异生物相容性和水溶性的材料制成。海藻糖和透明质酸 （HA） 作为尖端材料，为外泌体提供保护，选择溶解在无水乙醇中的聚乙烯吡咯烷酮 （PVP） 作为基材。使用显微镜和扫描电子显微镜 （SEM） 对微针贴片的形态进行了表征。使用拉伸仪评估微针的机械测试，以确认其穿透皮肤的能力，并在猪皮肤上的释放速率为 60 s。此外，使用透射电子显微镜 （TEM） 、纳米颗粒示踪分析 （NTA） 和蛋白质印迹 （WB） 对新鲜外泌体和exo@MN中外泌体的形态、大小和蛋白质含量进行了表征。使用共聚焦激光扫描显微镜 （CLSM） 表征树突状细胞 （DC） 对外泌体的内化，并通过流式细胞术评估 DC 的成熟。两种外泌体的形态学特征和生物学功能基本一致。

<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

外泌体作为新兴的“下一代”生物治疗药物和药物递送载体，在从药物递送和再生医学到疾病诊断和肿瘤免疫治疗等多个生物医学领域具有巨大潜力。然而，传统推注的快速清除和外泌体的稳定性差限制了其临床应用。微针作为一种溶液，可延长外泌体在给药部位的停留时间，从而维持药物浓度并促进持续的治疗效果。此外，微针还具有维持生物活性物质稳定性的能力。因此，我们介绍了一种用于加载和递送外泌体的微针贴片，并分享了包括外泌体分离、制造和表征外泌体负载的微针贴片的方法。以海藻糖和透明质酸为尖端材料，聚乙烯吡咯烷酮为基材，通过两步铸造法制备微针贴片。微针表现出强大的机械强度，尖端能够承受 2 N。猪皮用于模拟人体皮肤，微针尖端在皮肤穿刺后 60 秒内完全熔化。从微针释放的外泌体表现出与新鲜外泌体相当的形态、粒径、标志蛋白和生物学功能，使树突状细胞能够摄取并促进其成熟。

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.

作者聚焦：用于增强外泌体递送和稳定性的创新微针策略

用于加载和递送外泌体的微针贴片的制造和表征

My research focused on the efficient development of exosomes, the development of the microneedle strategy can increase exsome stability, enhance provide more possibility for the clinical application of exosomes. We will successfully develop the for advanced microneedle delivery systems. Those include self-healing porous microneedles for sustained drug delivery, ultrasound microneedle device for rapid loading and delivery of drugs, and cryomicroneedles for delivery of living cell, and sugar microneedles for delivery of exosomes.We use microneedle to pierce the biological barrier and then deliver exocells directly to the vector area efficiently. By storing them dry, we enhance their stability, making them precise, easy, and convenient for both clinical and home use. My lab, we focused on key technologies for developing the advanced microneedle-based delivery systems and that can load and deliver various therapeutics.Includes MRA, small molecular drug, antibodies, cell, and exosome. We are also investigate their chemical and physical properties and therapeutic efficacy in different biomedical applications.

在这里，我们提出了一种用于分离外泌体、exo@MN 贴片的制造和表征的方案。 图 1 说明了制造exo@MN贴片的工艺流程图。将外泌体与海藻糖和 HA 混合，然后将混合物加入微针模具中并离心分离。这个过程促进了外泌体在针尖的聚集，促进了快速释放。干燥后，加入 PVP 溶液并离心以完全填充模具。完全干燥后，去除霉菌以获得exo@MN补丁。使用显微镜和 SEM 表征微针的形态，如图...

外泌体具有显著的临床潜力， 但由于体内易 清除且稳定性差，其实际应用受到限制。微针提供了一种解决方案，通过刺穿生理屏障和干态保存来实现局部递送，从而解决了外泌体给药的局限性并扩大了其临床应用。

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

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.

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.

科研

生物工程

Isolation of Exosomes from Mouse Ovarian Epithelial Cancer Cells for Generating Microneedle Patch

To begin, take cultured mouse ovarian epithelial cancer cells. Remove the culture medium from the Petri dishes and wash twice with DPBS. Add 20 milliliters of Dulbecco's Modified Eagle Medium without FBS and penicillin-streptomycin solution.Incubate at 37 degrees Celsius and 5%carbon dioxide for 48 hours. Then collect the cell culture supernatant from 20 Petri dishes of 15-centimeter diameter. Centrifuge at 300 G for 10 minutes at four degrees Celsius and collect the supernatant.Centrifuge the supernatant at 2, 000 G for 10 minutes at four degrees Celsius, and collect the supernatant to remove cellular debris. Now turn on the vacuum pump and connect it to the air inlet of the vacuum filtration system. Filter the supernatant through the 0.22-micron polyethersulfone membrane to remove vesicles or impurities larger than 0.22 microns.Add 15 milliliters of the filtered supernatant to the inner tube of the 100-kilodalton ultrafiltration tube. Centrifuge at 3, 500 G for 10 minutes at four degrees Celsius. Then remove the liquid from the outer tube.Next, collect the liquid from the inner tube. Add one milliliter of DPBS to the inner tube. Pipette repeatedly to resuspend exosomes, and then collect them.Centrifuge the liquid at 10, 000 G for 60 minutes at four degrees Celsius. Transfer the supernatant to a six-milliliter quick-snap centrifuge tube and seal it with a heat sealer. Cut open the tube after ultracentrifuging the supernatant for two hours.Once the supernatant is removed, resuspend the pellet in 100 microliters of DPBS to obtain the exosome solution.

从小鼠卵巢上皮癌细胞中分离外泌体以生成微针贴剂

Fabrication of Microneedle Using Exosomes Isolated from Mouse Ovarian Epithelial Cancer Cells

To begin, mix components A and B of polydimethylsiloxane, or PDMS, in a ratio of 10 to one. After stirring the mixture well, pour the mixture into a master mold. Place the PDMS mold under a pressure of one pound per square inch for 15 minutes to remove air bubbles.Cure the PDMS mold at 60 degrees Celsius for two hours. De-mold to obtain the production mold of the microneedle. Then, clean the production mold with ultra pure water before use.Quantify the protein content of the prepared exosome solution using a BCA assay kit. Add an appropriate amount of DPBS to the exosome solution. Now, prepare a solution of 200 milligrams per milliliter of trehalose using DPBS as the solvent.Stir for 20 minutes to ensure full dissolution. Next, prepare a solution of hyaluronic acid and polyvinylpyrrolidone, or PVP, using DPBS and ethanol as the solvent, respectively. Stir overnight to ensure full dissolution.Mix the exosome solution and trehalose solution in an equal ratio. Add an equal volume of hyaluronic acid solution and mix thoroughly to obtain the tip solution. Using a plasma cleaner, process the surface of the PDMS mold for 30 seconds at a low grade to enhance its hydrophilicity.Add 40 microliters of the tip solution to the PDMS mold. Centrifuge at 3, 000G for three minutes at room temperature to ensure the liquid fills the needle layer of the mold. Remove excess liquid from the mold and dry at room temperature in a vacuum drying oven for one day.Then, add 200 microliters of PVP solution to the PDMS mold. Centrifuge at 3, 000G for three minutes at room temperature to ensure the liquid fills the mold's backing layer. After drying the mold for one day, de-mold to obtain the exosome-loaded microneedle's patch.

使用从小鼠卵巢上皮癌细胞中分离的外泌体制备微针

Morphological and Mechanical Characterization of Exosome Loaded Microneedle Patches

To begin, prepare the exosome loaded microneedle patch and paste its backing onto a 45-degree inclined surface. Position the patch under a stereo microscope. Turn on the illuminator and capture the morphology of the patch using a four times objective lens.For mechanical testing, cut out a three-by-three array from the patch and place it with the tips facing upwards on the rigid platform of a tensile meter. Adjust the height of the probe to bring it close to the needle tips without touching them. Set the parameters to stop automatically when the pressure reaches 20 Newton.Compress the needle tips vertically at a 0.5 millimeter per minute speed before recording the load versus displacement profile. The tips in the exosome loaded microneedle patch exhibited a conical shape arranged in a 10-by-10 array with sharp and intact needle tips. The tip of the microneedle can withstand the force of two Newtons, allowing it to penetrate through the skin barrier.