FLQ, Zhejiang Öncü Ar-Ge Programı (2022C03031), Çin Ulusal Anahtar Araştırma ve Geliştirme Programı (2021YFA0910103), Çin Ulusal Doğa Bilimleri Vakfı (22274141, 22074080), Shandong Eyaleti Doğa Bilimleri Vakfı (ZR2022ZD28) ve Shandong Eyaleti Taishan Burs Programı (tsqn201909106) tarafından desteklenen desteği takdir etmektedir. HC, Çin Ulusal Doğa Bilimleri Vakfı'nın (82202329) mali desteğini kabul eder. Yazarlar, Çin Bilimler Akademisi, Hangzhou Tıp Enstitüsü'ndeki (HIM) Paylaşılan Enstrümantasyon Çekirdek Tesisinde cihazların kullanımını kabul etmektedir.

Bölüm 3'te açıklanan deneyler için kullanılan domuz derisi, deney hayvanlarından elde edilmediği için piyasadan yenilebilir domuz kulakları olarak satın alındığından, bu çalışma etik izin gerektirmez.
1. Eksozomların izolasyonu
<ol><li>Hücre kültürü<ol><li>%10 fetal sığır serumu ve %1 penisilin-streptomisin solüsyonu (100×) içeren Dulbecco'nun Modifiye Eagle Medium (DMEM) kültür ortamında fare yumurtalık epitel kanseri hücreleri ID8'i yetiş...

Biyomedikal Uygulamalarda Eksozomların Sürekli Verilmesi için Mikroiğne Yamaları

<ol>
	<li>Barile, L., Vassalli, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes:+Therapy+delivery+tools+and+biomarkers+of+diseases.">Exosomes: Therapy delivery tools and biomarkers of diseases.</a> Pharmacol Ther. 174, 63-78 (2017).</li><li>Kalluri, R., Lebleu, V. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biology,+function,+and+biomedical+applications+of+exosomes.">The biology, function, and biomedical applications of exosomes.</a> Science. 367 (640), eaau6977 (2020).</li><li>Mathieu, M., Martin-Jaular, L., Lavieu, G., Thery, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specificities+of+secretion+and+uptake+of+exosomes+and+other+extracellular+vesicles+for+cell-to-cell+communication.">Specificities of secretion and uptake of exosomes and other extracellular vesicles for cell-to-cell communication.</a> Nat Cell Biol. 21 (1), 9-17 (2019).</li><li>Nair, A., 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=Hybrid+nanoparticle+system+integrating+tumor-derived+exosomes+and+poly(amidoamine)+dendrimers:+Implications+for+an+effective+gene+delivery+platform.">Hybrid nanoparticle system integrating tumor-derived exosomes and poly(amidoamine) dendrimers: Implications for an effective gene delivery platform.</a> Chem Mater. 35 (8), 3138-3150 (2023).</li><li>Lu, Z., 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=Dendritic+cell-derived+exosomes+elicit+tumor+regression+in+autochthonous+hepatocellular+carcinoma+mouse+models.">Dendritic cell-derived exosomes elicit tumor regression in autochthonous hepatocellular carcinoma mouse models.</a> J Hepatol. 67 (4), 739-748 (2017).</li><li>Oskouie, M. N., Aghili Moghaddam, N. S., Butler, A. E., Zamani, P., Sahebkar, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+use+of+curcumin-encapsulated+and+curcumin-primed+exosomes.">Therapeutic use of curcumin-encapsulated and curcumin-primed exosomes.</a> J Cell Physiol. 234 (6), 8182-8191 (2019).</li><li>Yuan, 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=Macrophage+exosomes+as+natural+nanocarriers+for+protein+delivery+to+inflamed+brain.">Macrophage exosomes as natural nanocarriers for protein delivery to inflamed brain.</a> Biomaterials. 142, 1-12 (2017).</li><li>Liao, W., 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=Exosomes:+The+next+generation+of+endogenous+nanomaterials+for+advanced+drug+delivery+and+therapy.">Exosomes: The next generation of endogenous nanomaterials for advanced drug delivery and therapy.</a> Acta Biomater. 86, 1-14 (2019).</li><li>Zhu, X., 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=Comprehensive+toxicity+and+immunogenicity+studies+reveal+minimal+effects+in+mice+following+sustained+dosing+of+extracellular+vesicles+derived+from+hek293t+cells.">Comprehensive toxicity and immunogenicity studies reveal minimal effects in mice following sustained dosing of extracellular vesicles derived from hek293t cells.</a> J Extracell Vesicles. 6 (1), 1324730 (2017).</li><li>Wen, 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=Biodistribution+of+mesenchymal+stem+cell-derived+extracellular+vesicles+in+a+radiation+injury+bone+marrow+murine+model.">Biodistribution of mesenchymal stem cell-derived extracellular vesicles in a radiation injury bone marrow murine model.</a> Int J Mol Sci. 20 (21), 5468-5482 (2019).</li><li>Cheng, Y., Zeng, Q., Han, Q., Xia, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+pH,+temperature+and+freezing-thawing+on+quantity+changes+and+cellular+uptake+of+exosomes.">Effect of pH, temperature and freezing-thawing on quantity changes and cellular uptake of exosomes.</a> Protein Cell. 10 (4), 295-299 (2019).</li><li>Jana, B. A., Shivhare, P., Srivastava, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gelatin-PVP+dissolving+microneedle-mediated+therapy+for+controlled+delivery+of+nifedipine+for+rapid+antihypertension+treatment.">Gelatin-PVP dissolving microneedle-mediated therapy for controlled delivery of nifedipine for rapid antihypertension treatment.</a> Hypertens Res. 47 (2), 427-434 (2024).</li><li>Zheng, Y., 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=A+rapidly+dissolvable+microneedle+patch+with+tip-accumulated+antigens+for+efficient+transdermal+vaccination.">A rapidly dissolvable microneedle patch with tip-accumulated antigens for efficient transdermal vaccination.</a> Macromol Biosci. 23 (12), e2300253 (2023).</li><li>Huang, 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=Efficient+delivery+of+nucleic+acid+molecules+into+skin+by+combined+use+of+microneedle+roller+and+flexible+interdigitated+electroporation+array.">Efficient delivery of nucleic acid molecules into skin by combined use of microneedle roller and flexible interdigitated electroporation array.</a> Theranostics. 8 (9), 2361-2376 (2018).</li><li>Zhou, Z., 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=Reverse+immune+suppressive+microenvironment+in+tumor+draining+lymph+nodes+to+enhance+anti-pd1+immunotherapy+via+nanovaccine+complexed+microneedle.">Reverse immune suppressive microenvironment in tumor draining lymph nodes to enhance anti-pd1 immunotherapy via nanovaccine complexed microneedle.</a> Nano Res. 13 (6), 1509-1518 (2020).</li><li>Kim, Y. C., Park, J. H., Prausnitz, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microneedles+for+drug+and+vaccine+delivery.">Microneedles for drug and vaccine delivery.</a> Adv Drug Deliv Rev. 64 (14), 1547-1568 (2012).</li><li>Bui, V. 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>

Şu anda, eksozomları izole etmek için ana yöntemler arasında ultrasantrifüjleme, yoğunluk gradyanlı santrifüjleme, ultrafiltrasyon, çökeltme, immünoaffinite manyetik boncuklar ve mikroakışkanlar24 bulunmaktadır. Küçük iğne ucu boşluklarının neden olduğu mikro iğnelerin sınırlı yükleme kapasitesi nedeniyle, daha fazla yükleme yapmak için eksozom konsantrasyonunu artırmak gerekir. Bu nedenle, hücre kültürü süpernatantını konsantre etmek için ultrafiltrasyonu se...

Hücreler tarafından hücre dışı matrise salınan küçük veziküller olan eksozomlar, çeşitli hastalıkların ve kanserlerin tedavisi için potansiyel biyoterapötikler ve ilaç dağıtım vektörleri olarak önerilmiştir1. Biyogenez süreçleri sırasında eksozomlar, fonksiyonel proteinler ve nükleik asitler2 dahil olmak üzere hücrelerin içinden çeşitli biyolojik olarak aktif molekülleri kapsüller. Sonuç olarak, taşıma işlemi sırasında alıcı hücreler tarafından alındığında, eksozomlar hedef hücrelerde gen ekspresyonunu ve hücresel fonksiyonları modüle etme yeteneğine sahiptir3. Bir tür doğal bilgi habercisi olarak, eksozomlar doku yenilenmesinde, bağışıklık regülasyonunda ve bir dağıtım taşıyıcısı olarak tam olarak kullanılmıştır4. Mühendislik teknikleri sayesinde, eksozomların yüzeyinde spesifik ligandlar zenginleştirilebilir, bu da alıcı hücrelerde sinyal olaylarının indüksiyonunu veya inhibisyonunu mümkün kılarveya belirli hücre tiplerini hedefler5. Kemoterapötik ajanlar ayrıca kanser tedavisi için eksozomlara da yüklenebilir6. Ayrıca, eksozomlar, terapötik kargo teslimatı için kan-beyin bariyerini geçme yeteneğine sahiptir ve bu da onları beyin bozukluklarının tedavisi için oldukça umut verici hale getirir7. Lipozomlarla karşılaştırıldığında, eksozomlar gelişmiş hücresel alım ve gelişmiş biyouyumluluk sergiler8. Daha iyi tolerans ve daha düşük toksisite gösterirken diğer hücrelere verimli bir şekilde girebilirler9. Bununla birlikte, eksozomların geleneksel bolus enjeksiyonu, kan dolaşımındaki karaciğer, böbrekler ve dalak tarafından sekestrasyona ve hızlı temizlemeye eğilimlidir10. Ayrıca, eksozomlar in vitro olarak zayıf stabiliteye sahiptir ve klinik uygulamalarını kısıtlayan depolama koşullarına karşı hassastır11.
Bir dizi mikrometrik boyutlu iğne ucu olan mikroiğneler, küçük moleküllü ilaçların12, proteinlerin13, nükleik asitlerin14 ve nanoilaçların15 verilmesi için fizyolojik engellere nüfuz etme yeteneğine sahiptir. Mikroiğneler, cilt yüzeyindeki lezyonları hedeflemek için hassas bir şekilde tasarlanmıştır ve dağınık uçları, hedeflenen bölgede homojen ilaç dağılımı sağlar, böylece terapötik etkilerini artırır16. Mikroiğnelerin tasarımı ve malzeme bileşimi, proteinler ve nükleik asitler gibi biyoaktif maddelerin kuru depolanmasını kolaylaştırarak stabilitelerini arttırır17. Geleneksel enjeksiyon yöntemleri nispeten kısa bir etki süresine sahiptir ve ağrıya neden olarak hastalarda korkuya neden olabilir18. Mikrometre boyutundaki mikroiğne uzunluğu, doku travmasını en aza indirir ve sinir uyarımını önler, böylece ağrıyı ortadan kaldırır ve hasta uyumunu artırır19. Ek olarak, mikroiğnelerin kullanıcı dostu yapısı, hastaların uzman personele ihtiyaç duymadan tedaviyi kendi kendilerine uygulamalarına olanak tanır16. Mikroiğneler cildin yanı sıra gözler20, ağız mukozası21, kalp22 ve kan damarları23 gibi dokularda da kullanılabilir. Eksozomların klinik olarak verilmesi için mikroiğnelerin uygulanması umut verici ve ileriye dönük bir strateji sağlar.
Bu nedenle, eksozom yüklü bir mikroiğne (exo@MN) yaması tanıtıyoruz ve üretim yöntemini açıklıyoruz. Mikroiğne yamaları, mikroiğne uçlarında eksozomların toplanmasını teşvik eden ve böylece dağıtım verimliliğini artıran santrifüjleme ve vakumlu kurutma ile birlikte iki aşamalı bir döküm yöntemi kullanılarak üretildi. Hem iğne uçları hem de destek, mükemmel biyouyumluluk ve suda çözünürlük sergileyen malzemeler kullanılarak yapılmıştır. Eksozomlar için koruma sağlamak için trehaloz ve hyaluronik asit (HA) uç materyaller olarak dahil edildi ve destek materyali olarak mutlak etanol içinde çözünen polivinilpirolidon (PVP) seçildi. Mikroiğne yamasının morfolojisi, mikroskopi ve taramalı elektron mikroskobu (SEM) kullanılarak karakterize edildi. Mikroiğnenin mekanik testi, cilde nüfuz etme yeteneklerini doğrulamak için bir gerilme ölçer kullanılarak değerlendirildi ve domuz derisi üzerindeki salım hızının 60 s olduğu araştırıldı. Ayrıca, exo@MN'daki hem taze eksozomların hem de eksozomların morfolojisi, boyutu ve protein içeriği, transmisyon elektron mikroskobu (TEM), nanopartikül izleme analizi (NTA) ve western blotting (WB) kullanılarak karakterize edildi. Eksozomların dendritik hücreler (DC'ler) tarafından içselleştirilmesi, konfokal lazer tarama mikroskobu (CLSM) kullanılarak karakterize edildi ve DC'lerin olgunlaşması akış sitometrisi ile değerlendirildi. İki tür eksozomun morfolojik karakterizasyonu ve biyolojik işlevleri esasen tutarlıdır.

<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

Ortaya çıkan "yeni nesil" biyoterapötikler ve ilaç dağıtım vektörleri olarak eksozomlar, ilaç dağıtımı ve rejeneratif tıptan hastalık teşhisi ve tümör immünoterapisine kadar çeşitli biyomedikal alanlarda muazzam bir potansiyele sahiptir. Bununla birlikte, geleneksel bolus enjeksiyonu ile hızlı klerens ve eksozomların zayıf stabilitesi, klinik uygulamalarını kısıtlamaktadır. Mikroiğneler, eksozomların uygulama bölgesinde kalma süresini uzatan, böylece ilaç konsantrasyonunu koruyan ve sürekli terapötik etkileri kolaylaştıran bir çözelti görevi görür. Ek olarak, mikroiğneler ayrıca biyoaktif maddelerin stabilitesini koruma yeteneğine de sahiptir. Bu nedenle, eksozomların yüklenmesi ve iletilmesi için bir mikroiğne yaması sunuyoruz ve eksozomların izolasyonu, eksozom yüklü mikroiğne yamalarının üretimi ve karakterizasyonu dahil olmak üzere yöntemleri paylaşıyoruz. Mikroiğne yamaları, uç malzemeleri olarak trehaloz ve hyaluronik asit ve destek malzemesi olarak polivinilpirolidon kullanılarak iki aşamalı bir döküm yöntemi ile üretildi. Mikroiğneler, insan derisini simüle etmek için 2 N. Domuz derisine dayanabilen uçlarla sağlam mekanik mukavemet gösterdi ve mikroiğnelerin uçları cilt delinmesinden sonra 60 saniye içinde tamamen eridi. Mikroiğnelerden salınan eksozomlar, morfoloji, partikül boyutu, işaretleyici proteinler ve taze eksozomlarınkiyle karşılaştırılabilir biyolojik işlevler sergiledi, bu da dendritik hücrelerin alımını sağladı ve olgunlaşmalarını teşvik etti.

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.

Yazar Spotlight: Gelişmiş Eksozom İletimi ve Stabilitesi için Yenilikçi Mikroiğne Tabanlı Stratejiler

Eksozomların yüklenmesi ve teslimi için mikroiğne yamalarının üretimi ve karakterizasyonu

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.

Burada, eksozomların izolasyonu, exo@MN yamanın üretimi ve karakterizasyonu için bir protokol sunuyoruz. Şekil 1 , exo@MN yamanın üretimi için süreç akış şemasını göstermektedir. Eksozomlar trehaloz ve HA ile karıştırıldı ve karışım daha sonra mikroiğne kalıbına eklendi ve santrifüjlendi. Bu işlem, eksozomların iğne uçlarında toplanmasını kolaylaştırarak hızlı salınımı teşvik etti. Kuruduktan sonra, PVP çözeltisi ilave edildi ve kalıbı tamamen d...

Eksozomlar önemli klinik potansiyele sahiptir, ancak kolay in vivo klerens ve zayıf stabilite nedeniyle pratik uygulamaları sınırlıdır. Mikroiğneler, fizyolojik engelleri delerek ve kuru halde koruma sağlayarak lokalize uygulamayı mümkün kılarak, böylece eksozom uygulamasının sınırlamalarını ele alarak ve klinik faydalarını genişleterek bir çözüm sunar.

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

561 Views.  Tianjin University. 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 mi...

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

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.

Biyomühendislik

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.

Mikroiğne Yaması Oluşturmak için Fare Yumurtalık Epitel Kanseri Hücrelerinden Eksozomların İzolasyonu

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.

Fare Yumurtalık Epitel Kanseri Hücrelerinden İzole Edilen Eksozomlar Kullanılarak Mikroiğne Üretimi

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.