Name: predicting gene silencing through the spatiotemporal control of sirna release from photo-responsive polymeric nanocarriers
Uploaded: 2017-07-21T15:00:00
Description: Watch this Scientific Journal Video about Predicting Gene Silencing Through the Spatiotemporal Control of siRNA Release from Photo-responsive Polymeric Nanocarriers at JoVE.com

Forfatterne takker National Institute of General Medical Sciences af National Institutes of Health (NIH) for økonomisk støtte gennem en institutionel udviklingspris (IDeA) under tildelingsnummer P20GM103541 samt tildelingsnummer P20GM10344615. Udtalelserne heri afspejler ikke NIH&#39;s synspunkter. Vi anerkender også Delaware Biotechnology Institute (DBI) og Delaware Economic Development Office (DEDO) for økonomisk støtte gennem Bioscience Center for Advanced Technology (Bioscience CAT) -prisen (12A00448).

1. Formulering af siRNA nanocarrierer <ol><li> Forbered separate opløsninger af siRNA og mPEG- b -P (APNBMA) med lige mængder fortyndet i 20 mM 4- (2-hydroxyethyl) piperazin-1-ethansulfonsyre (HEPES) buffer ved pH 6,0. <ol><li> Tilsæt siRNA i en koncentration på 32 μg / ml til 20 mM HEPES-opløsning. BEMÆRK: siRNA var en ikke-målrettet, universel negativ kontrol sekvens; SiRNA&#39;en kan imidlertid være designet til at målrette mod ethvert gen af ​​interesse. </li><li> Opl...

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Der er et par trin i den metode, der er særlig kritisk. Ved formulering af nanocarriererne er rækkefølgen af ​​komponenttilsætning og blandingshastighed to vigtige parametre, der påvirker effekten 39 . Denne protokol kræver, at den kationiske komponent, mPEG- b -P (APNBMA), tilsættes til den anioniske komponent, siRNA, dråbevis under vortexing. Afhængig af det totale formuleringsvolumen tager denne blandeproces 3-6 s. For at teste om nanocarriererne blev dannet ordentligt, m...

Små interfererende RNA&#39;er (siRNA&#39;er) medierer post-transkriptionelt gensilring gennem en katalytisk RNAi-vej, der er yderst specifik, kraftig og skræddersyet til stort set ethvert målgen 1 . Disse lovende egenskaber har gjort det muligt for siRNA-terapeutika at udvikle sig i humane kliniske forsøg til behandling af talrige sygdomme, herunder metastatisk melanom og hæmofili 2 , 3 . Imidlertid fortsætter betydelige leveringsproblemer, som har forhindret oversættelse 4 . Leveringskøretøjer skal især forblive stabile og beskytte siRNA&#39;er mod ekstracellulær nedbrydning, men frigive også nyttelasten i cytoplasma 5 . Desuden kræver mange RNAi-applikationer forbedrede fremgangsmåder til regulering af gentympning i rum og tid 6 , hvilket vil reducere bivirkninger i siRNA-terapeutik 7 og muliggøre transformativ aDvances i applikationer, der spænder fra celle-mikroarrays til lægemiddelforskning 8 til modulering af celleresponser i regenerative stilladser 9 . Disse udfordringer fremhæver behovet for nye materialer og metoder til bedre at kontrollere binding mod frigivelse i siRNA nanocarrier. En af de mest lovende strategier til styring af siRNA frigivelse og forbedring af spatiotemporal regulering er brugen af ​​stimuli-responsive materialer 10 . For eksempel er en bred vifte af biomaterialer blevet konstrueret med foranderlig nukleinsyrebindingsaffinitet som reaktion på ændret redoxpotentiale eller pH eller anvendte magnetfelter, ultralyd eller lys 11 . Selv om mange af disse systemer viser forbedret kontrol over nukleinsyreaktivitet, er anvendelsen af ​​lys som en trigger særligt fordelagtig på grund af dens øjeblikkelige tidsmæssige respons, præcis rumlig opløsning og nem afstemning <suP class = &quot;xref&quot;&gt; 12. Desuden er potentialet ved fotoknitiske teknologier til regulering af genekspression blevet påvist ved hjælp af state-of-the-art inducerbare promotor og optogenetiske regulatorsystemer; Imidlertid lider disse systemer af mange udfordringer, herunder begrænset kapacitet til at regulere endogene gener, sikkerhedsproblemer som immunogenicitet og vanskeligheder med at levere multikomponent-samlinger 13 , 14 , 15 . Foto-responsive siRNA nanocarrier er ideelt egnet til at overvinde disse ulemper og tilvejebringe en enklere og mere robust tilgang til spatiotemporalt modulere genekspression 16 , 17 , 18 . Desværre forbliver metoder til nøjagtigt at forudsige det resulterende proteinknockdown-svar uvæsentligt. En vigtig udfordring er, at kvantitative evalueringer af siRNA-udgivelsen erSjældne 19 , 20 , og selv når disse evalueringer udføres, er de ikke blevet koblet til analyser af siRNA / proteinomsætningsdynamik. Både mængden af ​​frigivet siRNA og dens persistens / levetid er vigtige determinanter for den resulterende gendæmpningsdynamik; En mangel på sådanne oplysninger er derfor en stor afbrydelse, der udelukker nøjagtig forudsigelse af dosisrespons i RNAi 21 . Adressering af denne udfordring ville fremskynde formuleringen af ​​de relevante struktur-funktion forhold i nanocarrier og bedre informere biomaterialedesigner 22 . Desuden ville sådanne fremgangsmåder muliggøre udvikling af mere effektive siRNA doseringsprotokoller. I et forsøg på at forstå det dynamiske lydsvarssvar har flere grupper undersøgt matematiske modeller af RNAi 23 , 24 , 25 . Disse rammer varSucces med at give indsigt i siRNA-medierede ændringer i genekspression og identificere hastighedsbegrænsende trin 26 . Imidlertid er disse modeller kun blevet anvendt til kommercielle genleveringssystemer ( fx lipofektamin og polyethylenimin (PEI)), der ikke er i stand til kontrolleret siRNA-frigivelse, og kompleksiteten af ​​modellerne har alvorligt begrænset deres anvendelighed 27 . Disse mangler fremhæver et ubehøvet behov for nye materialer, der er i stand til præcist indstillelig siRNA-frigivelse kombineret med strømlinede og nemme at bruge prædiktive kinetiske modeller. Vores metode behandler alle disse udfordringer gennem integration af en lysfølsom nanocarrier platform med koblede metoder til at kvantificere fri siRNA og model RNAi dynamik. Specielt overvåges vores platforms præcist styrede siRNA-udgivelse 28 ved hjælp af to komplementære metoder til nøjagtigt kvantificering af indkapslede kontra uBundet siRNA. De eksperimentelle data fra disse analyser indføres i en simpel kinetisk model for at forudsige genlydningseffektiviteter a priori 29 . Endelig udnyttes nanokarrierernes on / off-natur let for at frembringe cellemønstre i genekspression med rumlig kontrol på cellelængde skalaen. Således tilvejebringer denne metode en let tilpasningsbar metode til styring og forudsigelse af gentympning i en række forskellige applikationer, der ville have gavn af spatiotemporal regulering af celleadfærd.

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			<td height="42" style="height:42px;width:183px;">siRNA</td>
			<td style="width:157px;">Sigma-Aldrich</td>
			<td style="width:145px;">SIC001</td>
			<td style="width:195px;">non-targeted, universal negative control</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">mPEG-b-P(APNBMA)</td>
			<td style="width:157px;">synthesized in our lab</td>
			<td style="width:145px;">N/A</td>
			<td style="width:195px;">photo-responsive polymer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">HEPES</td>
			<td style="width:157px;">Fisher Scientific</td>
			<td style="width:145px;">BP310-100</td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">sodium dodecyl sulfate&#160;</td>
			<td style="width:157px;">Sigma-Aldrich</td>
			<td style="width:145px;">436143</td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">rubber gasket</td>
			<td style="width:157px;">McMaster-Carr</td>
			<td style="width:145px;">3788T21</td>
			<td style="width:195px;">0.5 mL thick</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:183px;">UV laser&#160;</td>
			<td style="width:157px;">Excelitas Technologies</td>
			<td style="width:145px;">Omnicure S2000</td>
			<td style="width:195px;">collimating lens and 365 nm filter used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">agarose</td>
			<td style="width:157px;">Fisher Scientific</td>
			<td style="width:145px;">BP160-100</td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">ethidium bromide</td>
			<td style="width:157px;">Fisher Scientific</td>
			<td style="width:145px;">BP1302-10</td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:183px;">siRNA labelled with Dy547</td>
			<td style="width:157px;">GE Healthcare Dharmacon, Inc.</td>
			<td style="width:145px;">custom order</td>
			<td style="width:195px;">fluorophore conjugated to 5&#8217; end of sense strand</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">microscope slide</td>
			<td style="width:157px;">Fisher Scientific</td>
			<td style="width:145px;">12-550-A3</td>
			<td style="width:195px;">pre-cleaned glass</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">Secure-Seal Spacer</td>
			<td style="width:157px;">Life Technologies</td>
			<td style="width:145px;">S24735</td>
			<td style="width:195px;">double-sided adhesive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">LSM 780&#160;</td>
			<td style="width:157px;">Carl Zeiss</td>
			<td style="width:145px;">N/A</td>
			<td style="width:195px;">confocal microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">ZEN 2010</td>
			<td style="width:157px;">Carl Zeiss</td>
			<td style="width:145px;">N/A</td>
			<td style="width:195px;">FCS analysis software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">MATLAB</td>
			<td style="width:157px;">MathWorks</td>
			<td style="width:145px;">N/A</td>
			<td style="width:195px;">programming language</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">NIH/3T3 cells&#160;</td>
			<td style="width:157px;">ATCC</td>
			<td style="width:145px;">ATCC CRL-1658</td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">DMEM</td>
			<td style="width:157px;">Mediatech</td>
			<td style="width:145px;">10-013-CV</td>
			<td style="width:195px;">growth media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">fetal bovine serum</td>
			<td style="width:157px;">Mediatech</td>
			<td style="width:145px;">35-011-CV</td>
			<td style="width:195px;">heat-inactivated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">penicillin-streptomycin</td>
			<td style="width:157px;">Mediatech</td>
			<td style="width:145px;">&#160;30-002-CI</td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">6-well plates</td>
			<td style="width:157px;">Fisher Scientific</td>
			<td style="width:145px;">08-772-1B</td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">Opti-MEM</td>
			<td style="width:157px;">Life Technologies</td>
			<td style="width:145px;">11058021</td>
			<td style="width:195px;">transfection media</td>
		</tr>
	</tbody>
</table>

predicting gene silencing through the spatiotemporal control of sirna release from photo-responsive polymeric nanocarriers

Nye materialer og metoder er nødvendige for bedre at kontrollere bindingen vs. frigivelse af nukleinsyrer til en lang række anvendelser, som kræver præcis regulering af genaktivitet. Navnlig ville nye stimuli-responsive materialer med forbedret spatiotemporal kontrol over genekspression åbne for oversættelige platforme i lægemiddelforskning og regenerativ medicinteknologi. Desuden ville en forøget evne til at styre nukleinsyrefrigivelse basis muliggøre udviklingen af strømlinede metoder til at forudsige Nanobærer virkningsfuldhed a priori, hvilket fører til fremskyndet screening af leveringsvehikler. Her præsenterer vi en protokol til forudsigelse af genlyddningseffektivitet og opnåelse af spatiotemporal kontrol over genekspression gennem et modulært fotoresvarende nanocarrier-system. Lille interfererende RNA (siRNA) komplekseres med mPEG- b- poly (5- (3- (amino) propoxy) -2-nitrobenzylmethacrylat) (mPEG- b -P (APNBMA)) polymerer tilRm stabile nanocarriers, der kan styres med lys for at lette afstemning, on / off siRNA release. Vi skitserer to komplementære analyser, der anvender fluorescenskorrelationsspektroskopi og gelelektroforese til den nøjagtige kvantificering af siRNA-frigivelse fra opløsninger, der efterligner intracellulære miljøer. Oplysninger opnået fra disse assays blev inkorporeret i en simpel RNA interferens (RNAi) kinetisk model for at forudsige de dynamiske afstødningsresponser på forskellige foto-stimulusbetingelser. Til gengæld tillod disse optimerede bestrålingsbetingelser forfining af en ny protokol til spatiotemporalt styring af gentympning. Denne metode kan generere cellulære mønstre i genekspression med celle til celleopløsning og ingen påviselige off-target-effekter. Tværtimod tilbyder vores tilgang en nem at bruge metode til at forudsige dynamiske ændringer i genekspression og præcis styring af siRNA-aktivitet i rum og tid. Dette sæt assays kan let tilpasses til at teste en bred vifte af otHendes stimuli-responsive systemer for at imødegå vigtige udfordringer, der er relevante for en lang række anvendelser inden for biomedicinsk forskning og medicin.

Efter formuleringen af ​​nanocarriererne blev der udført siRNA-frigivelsesassays for at informere bestrålingsbetingelserne, som skal anvendes i in vitro- transfektionerne. Forskellige doser af lys blev påført for at bestemme procenten af ​​siRNA, der blev frigivet. Det første assay anvendte gelelektroforese til adskillelse af de frie siRNA-molekyler fra siRNA-molekylerne, der stadig er kompleksbundet / associeret med polymeren. Nanocarrier, der ikke blev behandlet m...

Watch this Scientific Journal Video about Predicting Gene Silencing Through the Spatiotemporal Control of siRNA Release from Photo-responsive Polymeric Nanocarriers at JoVE.com

Predicting Gene Silencing Through the Spatiotemporal Control of siRNA Release from Photo-responsive Polymeric Nanocarriers

Vi præsenterer en ny metode, der bruger fotobesvarende blokcopolymerer til mere effektiv spatiotemporal styring af gentympning uden påviselige off-target-effekter. Endvidere kan ændringer i genekspression forudsiges ved anvendelse af straightforward siRNA frigivelsesassays og simpel kinetisk modellering.

Forudsigelse af gensvulmning gennem spatiotemporal kontrol af siRNA-frigivelse fra fotoresvarende polymere nanocarriers

New materials and methods are needed to better control the binding vs. release of nucleic acids for a wide range of applications that require the precise ...

Research

JoVE Journal

Bioengineering

Formulation of Diblock Polymeric Nanoparticles through Nanoprecipitation Technique

Nanotechnology is a relatively new branch of science that involves harnessing the unique properties of particles that are nanometers in scale ...

Combinatorial Synthesis of and High-throughput Protein Release from Polymer Film and Nanoparticle Libraries

Kombinatorisk syntese af og Højkapacitetsforskning Protein Udgivelse fra Polymer Film og nanopartikel Biblioteker

Polyanhydrides are a class of biomaterials with excellent biocompatibility and drug delivery capabilities. While they have been studied extensively with ...

Engineering a Bilayered Hydrogel to Control ASC Differentiation

Bearbejdning af et dobbeltlag Hydrogel at styre ASC differentiering

Natural polymers over the years have gained more importance because of their host biocompatibility and ability to interact with cells in vitro and in ...

Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry

Evaluering af Polymeric Gene Delivery nanopartikler ved Nanopartikel Tracking Analyse og Højkapacitetsforskning Flowcytometri

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a ...

The Use of the Ex Vivo Chandler Loop Apparatus to Assess the Biocompatibility of Modified Polymeric Blood Conduits

The Use of the Ex Vivo Chandler Loop Apparatus to Assess the Biocompatibility of Modified Polymeric Blood Conduits

Brug af den<em&gt; Ex vivo</em&gt; Chandler Loop Apparat til Vurdere biokompatibilitet modificerede Polymere Blood Kanaler

The foreign body reaction occurs when a synthetic surface is introduced to the body. It is characterized by adsorption of blood proteins and the ...

Porous Silicon Microparticles for Delivery of siRNA Therapeutics

Porøst silicium mikropartikler til levering af siRNA Therapeutics

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

Polymeric Microneedle Array Fabrication by Photolithography

Polymere mikronålarray Fabrication ved Fotolitografi

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

Fabricating Superhydrophobic Polymeric Materials for Biomedical Applications

Opdigte superhydrofobe Polymere materialer til biomedicinske anvendelser

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial ...

Synthesis of Gold Nanoparticle Integrated Photo-responsive Liposomes and Measurement of Their Microbubble Cavitation upon Pulse Laser Excitation

Syntese af Gold Nanopartikel Integrated Foto-responsive Liposomer og måling af deres Mikroboble Kavitation på Pulse Laser Excitation

Photo-responsive nanoparticles (NPs) have received considerable attention because of their potential in providing spatial, temporal, and dosage control ...

Delivery of Therapeutic siRNA to the CNS Using Cationic and Anionic Liposomes

Levering af terapeutisk siRNA til CNS Brug kationiske og anioniske liposomer

Prion diseases result from the misfolding of the normal, cellular prion protein (PrPC) to an abnormal protease resistant isomer called PrPRes.  The ...