Name: Auteur in de schijnwerpers: Innovatieve strategieën op basis van micronaalden voor verbeterde exosoomafgifte en stabiliteit
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. waardeert de steun van het Pioneer R&D-programma van Zhejiang (2022C03031), het National Key Research and Development Program van China (2021YFA0910103), de National Natural Science Foundation of China (22274141, 22074080), de Natural Science Foundation van de provincie Shandong (ZR2022ZD28) en het Taishan Scholar Program van de provincie Shandong (tsqn201909106). H.C. erkent de financiële steun van de National Natural Science Foundation of China (82202329). De auteurs erkennen het gebruik van instrumenten in de Shared Instrumentation Core Facility van het Hangzhou Institute of Medicine (HIM), Chinese Academie van Wetenschappen.

Voor dit onderzoek is geen ethische goedkeuring vereist, aangezien de varkenshuid die voor de in sectie 3 beschreven experimenten is gebruikt, als eetbare varkensoren op de markt is gekocht en niet afkomstig is van proefdieren.
1. Isolatie van exosomen
<ol><li>Celcultuur<ol><li>Kweek ovariumepitheelcellen ID8 van muizen in Dulbecco's Modified Eagle Medium (DMEM) kweekmedium met 10% foetaal runderserum en 1% penicilline-streptomycine-oplossing (100×) (zie Materiaalta...

Micronaaldpleisters voor langdurige afgifte van exosomen in biomedische toepassingen

<ol>
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Momenteel zijn de belangrijkste methoden voor het isoleren van exosomen ultracentrifugatie, dichtheidsgradiëntcentrifugatie, ultrafiltratie, precipitatie, immunoaffiniteit magnetische kralen en microfluïdica24. Vanwege de beperkte laadcapaciteit van micronaalden, veroorzaakt door hun kleine naaldpuntruimte, is het noodzakelijk om de concentratie van exosomen te verhogen om meer te laden. Daarom kozen we voor ultrafiltratie om het supernatans van de celcultuur te concentreren en gebruikten we ver...

Exosomen, kleine blaasjes die door cellen in de extracellulaire matrix worden afgegeven, zijn voorgesteld als potentiële biotherapeutica en vectoren voor medicijnafgifte voor de behandeling van verschillende ziekten enkankers. Tijdens hun biogeneseproces kapselen exosomen verschillende biologisch actieve moleculen in de cellen in, waaronder functionele eiwitten en nucleïnezuren2. Als gevolg hiervan hebben exosomen, wanneer ze tijdens het transportproces door ontvangende cellen worden opgenomen, het vermogen om genexpressie en cellulaire functies in de doelcellen te moduleren3. Als een soort natuurlijke informatieboodschapper zijn exosomen ten volle benut bij weefselregeneratie, immuunregulatie en als drager van de bevalling4. Door middel van technische technieken kunnen specifieke liganden worden verrijkt op het oppervlak van exosomen, waardoor de inductie of remming van signaleringsgebeurtenissen in ontvangende cellen mogelijk wordt of gericht op specifieke celtypen5. Chemotherapeutische middelen kunnen ook in exosomen worden geladen voor de behandeling van kanker6. Bovendien hebben exosomen het vermogen om de bloed-hersenbarrière te passeren voor therapeutische ladingafgifte, waardoor ze veelbelovend zijn voor de behandeling vanhersenaandoeningen. Vergeleken met liposomen vertonen exosomen een verbeterde cellulaire opname en verbeterde biocompatibiliteit8. Ze zijn in staat om efficiënt andere cellen binnen te dringen, terwijl ze een betere tolerantie en lagere toxiciteit vertonen9. De traditionele bolusinjectie van exosomen is echter vatbaar voor sekwestratie en snelle klaring door de lever, nieren en milt in de bloedbaan10. Bovendien hebben exosomen een slechte stabiliteit in vitro en zijn ze vatbaar voor opslagomstandigheden, die hun klinische toepassingen beperken11.
Micronaalden, een reeks naaldpunten van micrometrische grootte, hebben het vermogen om fysiologische barrières te doorbreken voor de afgifte van geneesmiddelen met kleine moleculen12, eiwitten13, nucleïnezuren14 en nanomedicijnen15. Micronaalden zijn nauwkeurig ontworpen om laesies op het huidoppervlak aan te pakken, en hun verspreide uiteinden zorgen voor een uniforme verdeling van het geneesmiddel op de beoogde plaats, waardoor hun therapeutische impact wordt versterkt16. Het ontwerp en de materiaalsamenstelling van micronaalden vergemakkelijken de droge opslag van bioactieve stoffen zoals eiwitten en nucleïnezuren, waardoor hun stabiliteit wordt verbeterd17. Traditionele injectiemethoden hebben een relatief korte werkingsduur en kunnen pijn veroorzaken, waardoor angst bij patiënten wordt opgewekt18. De lengte van de micronaald ter grootte van een micrometer minimaliseert weefseltrauma en voorkomt zenuwstimulatie, waardoor pijn wordt geëlimineerd en de therapietrouw van de patiënt wordtverbeterd19. Bovendien stelt het gebruiksvriendelijke karakter van micronaalden patiënten in staat om de behandeling zelf toe te dienen zonder dat er gespecialiseerd personeel nodigis 16. Naast de huid kunnen micronaalden ook worden gebruikt in weefsels zoals de ogen20, mondslijmvlies21, hart22 en bloedvaten23. De toepassing van micronaalden voor de klinische toediening van exosomen biedt een veelbelovende en prospectieve strategie.
Daarom introduceren we een exosoom-geladen micronaald (exo@MN) patch en onthullen we de fabricagemethode. De micronaaldpleisters werden vervaardigd met behulp van een gietmethode in twee stappen, samen met centrifugeren en vacuümdrogen, wat de aggregatie van exosomen aan de uiteinden van de micronaald bevordert, waardoor de afgifte-efficiëntie wordt verbeterd. Zowel de naaldpunten als de achterkant zijn gemaakt van materialen die een uitstekende biocompatibiliteit en oplosbaarheid in water vertonen. Trehalose en hyaluronzuur (HA) werden opgenomen als tipmaterialen om bescherming te bieden voor de exosomen, en polyvinylpyrrolidon (PVP) opgelost in absolute ethanol werd gekozen als dragermateriaal. De morfologie van de micronaaldpleister werd gekarakteriseerd met behulp van microscopie en scanning-elektronenmicroscoop (SEM). De mechanische tests van de micronaald werden beoordeeld met behulp van een trekmeter om hun vermogen om de huid binnen te dringen te bevestigen, en de afgiftesnelheid op varkenshuid werd onderzocht op 60 s. Bovendien werden de morfologie, grootte en eiwitinhoud van zowel verse exosomen als exosomen in exo@MN gekarakteriseerd met behulp van transmissie-elektronenmicroscoop (TEM), nanodeeltjesvolganalyse (NTA) en western blotting (WB). De internalisatie van exosomen door dendritische cellen (DC's) werd gekarakteriseerd met behulp van confocale laserscanmicroscoop (CLSM) en de rijping van DC's werd geëvalueerd door middel van flowcytometrie. De morfologische karakterisering en biologische functies van de twee soorten exosomen zijn in wezen 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

Exosomen, als opkomende biotherapeutica en vectoren voor de toediening van geneesmiddelen van de "volgende generatie", hebben een enorm potentieel op diverse biomedische gebieden, variërend van medicijnafgifte en regeneratieve geneeskunde tot ziektediagnose en tumorimmunotherapie. De snelle klaring door traditionele bolusinjectie en de slechte stabiliteit van exosomen beperken echter hun klinische toepassing. Micronaalden dienen als een oplossing die de verblijftijd van exosomen op de toedieningsplaats verlengt, waardoor de geneesmiddelconcentratie behouden blijft en aanhoudende therapeutische effecten worden vergemakkelijkt. Bovendien hebben micronaalden ook het vermogen om de stabiliteit van bioactieve stoffen te behouden. Daarom introduceren we een micronaaldpleister voor het laden en afleveren van exosomen en delen we de methoden, waaronder isolatie van exosomen, fabricage en karakterisering van met exosomen beladen micronaaldpleisters. De micronaaldpleisters werden vervaardigd met behulp van trehalose en hyaluronzuur als tipmaterialen en polyvinylpyrrolidon als rugmateriaal door middel van een gietmethode in twee stappen. De micronaalden vertoonden een robuuste mechanische sterkte, met tips die bestand waren tegen 2 N. Varkenshuid werd gebruikt om de menselijke huid te simuleren, en de uiteinden van micronaalden smolten volledig binnen 60 s na het doorboren van de huid. De exosomen die uit de micronaalden vrijkwamen, vertoonden morfologie, deeltjesgrootte, markereiwitten en biologische functies die vergelijkbaar waren met die van verse exosomen, waardoor dendritische cellen konden worden opgenomen en hun rijping werd bevorderd.

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.

Auteur in de schijnwerpers: Innovatieve strategieën op basis van micronaalden voor verbeterde exosoomafgifte en stabiliteit

Fabricage en karakterisering van micronaaldpleisters voor het laden en afleveren van exosomen

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.

Hier presenteren we een protocol voor de isolatie van exosomen, fabricage en karakterisering van exo@MN patch. Figuur 1 illustreert het processtroomschema voor de fabricage van exo@MN patch. De exosomen werden gemengd met trehalose en HA, en het mengsel werd vervolgens toegevoegd aan de micronaaldvorm en gecentrifugeerd. Dit proces vergemakkelijkte de aggregatie van exosomen aan de naaldpunten, waardoor een snelle afgifte werd bevorderd. Na het drogen werd PVP-oplossing toegevoegd en gecentr...

Exosomen hebben een aanzienlijk klinisch potentieel, maar hun praktische toepassing is beperkt vanwege de gemakkelijke in vivo klaring en slechte stabiliteit. Micronaalden bieden een oplossing door gelokaliseerde toediening mogelijk te maken door fysiologische barrières en conservering in droge toestand te doorboren, waardoor de beperkingen van exosoomtoediening worden aangepakt en hun klinische bruikbaarheid wordt uitgebreid.

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.

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.

Isolatie van exosomen uit ovariumepitheelkankercellen van muizen voor het genereren van een micronaaldpleister

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.

Fabricage van micronaald met behulp van exosomen geïsoleerd uit ovariumepitheelkankercellen van muizen

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.