Name: evaluation of polymeric gene delivery nanoparticles by nanoparticle tracking analysis and high-throughput flow cytometry
Uploaded: 2013-03-01T12:00:00
Description: Watch this Scientific Journal Video about Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry at JoVE.com

Forfatterne takker TEDCO MSCRF (2009-MSCRFE-0098-00) og NIH R21CA152473 for support.

1. Cellepodning <ol><li> Lad ikke cellerne til at vokse til overconfluency. Benytte tidlige passage-celler ved transfektion af primære celler. </li><li> Fireogtyve timer før transfektionen Trypsinisér cellerne, tæl cellerne under anvendelse af et hæmocytometer, og fortyndes cellesuspensionen med medier for at opnå den ønskede celledensitet (celler / volumen). Seed celler i klar vævskultur-behandlet fladbundede plader med 96 brønde under anvendelse af en beholder og multikanalspipetter. Den valgte massefylde b...

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Protokollerne ovenfor beskriver fremgangsmåder til evaluering af transfektion effekten af ​​nanopartikel formuleringer, såvel som en metode til karakterisering af partikelstørrelsen og DNA loading af nanopartiklerne. Antallet af plasmider pr partikel er en vigtig parameter, som kan hjælpe med at forudsige effektiviteten af ​​partiklen og kan også anvendes til fastsættelse af dosis. Nanopartikel sporing analyse kan udføres i en række forskellige vandige opløsninger, såsom de afviger i saltkoncentration....

Bestemmelse af antallet af plasmider kompleksbundet pr nanopartikel er vigtigt at udforme effektive nanopartikel-baserede genafgivelsessystemer strategier, navnlig til samtidig tilførsel af multiple plasmider i den samme celle målet, som det ofte er nødvendig stamcelle omprogrammering studier 12. Få metoder til at beregne antallet af plasmider, der er forbundet med en enkelt nanopartikel er blevet beskrevet, og hver metode har ulemper i de teknikker, der anvendes til estimering 13-16. Kvantepunktet (QD) mærkning kombineret med TEM er blevet anvendt til at estimere plasmider pr partikel i chitosan-baserede nanopartikler. Estimation med denne QD teknik er kompliceret på grund af behovet for at mærke DNA, hvilket kan ændre sine selvsamling egenskaber, muligheden for, at indkapslede umærket DNA ikke er umiddelbart detekteres; potentielt overlappende plasmider og QDs i 2D TEM-billeder af partikler, og andre forenklende antagelser 13. En alternativ fremgangsmåde, der er enpplicable når ordnet mikrodomæner findes i partiklerne er blevet anvendt til at undersøge lipopolyamin-DNA-komplekser via cryo-transmissionselektronmikroskopi (cryo-TEM), røntgenspredning, og dynamisk lysspredning (DLS) 14,15. Desværre materialer, såsom de polymere nanopartikler undersøgt her ikke anvendelse med denne metode. I en anden undersøgelse anvendes Collins et al en strøm partikel billedanalyse teknik til at studere (Lys) 16-indeholdende peptid / DNA-komplekser. Kan imidlertid deres metode kun evaluerer større, mikrometerstore partikler 16. Således har vi for nylig udviklet en ny og fleksibel assay til at kvantificere antallet af plasmider pr nanopartikel 11.

<table border="1"><tbody><tr><td></td><td></td><td></td><td> Reagens </td></tr><tr><td> Phosphatbufret saltvand, 1x (PBS) </td><td> Invitrogen </td><td> 10.010 </td><td></td></tr><tr><td> EGM-2mV BulletKit </td><td> Lonza </td><td> CC-3202 </td><td></td></tr><tr><td> Trypsin </td><td> Invitrogen </td><td> 25.300 </td><td></td></tr><tr><td> Natriumacetatbuffer </td><td> Sigma-Aldrich </td><td> S7899 </td><td> Fortyndes til 25 mM i deioniseret vand </td></tr><tr><td> Dimethylsulfoxid </td><td> Sigma-Aldrich </td><td> 276.855 </td><td></td></tr><tr><td> pEGFP DNA </td><td> Elim Biopharmaceuticals </td><td> NA </td><td></td></tr><tr><td> DsRed DNA </td><td> Addgene </td><td> 21.718 </td><td></td></tr><tr><td> PEI, forgrenet </td><td> Sigma-Aldrich </td><td> 408.727 </td> &lt;td&gt; </td></tr><tr><td> CellTiter 96 Aqueous One </td><td> Promega </td><td> G3580 </td><td></td></tr><tr><td></td><td></td><td></td><td> Materialer </td></tr><tr><td> Klar fladbundet 96-brønds plade, steril </td><td> Sarstedt </td><td> 82.1581.001 </td><td></td></tr><tr><td> Klar rundbundet 96-brønds plade, steril </td><td> Sarstedt </td><td> 82.1582.001 </td><td></td></tr><tr><td> 12-kanals Finnpipette </td><td> Thermo Scientific </td><td> NA </td><td> 5-50 og 50-300 ul </td></tr><tr><td> Fluorescensmikroskop </td><td> Zeiss </td><td> NA </td><td> Model nummer: AX10 </td></tr><tr><td> C6 Accuri flowcytometer </td><td> BD Biosciences </td><td> NA </td><td></td></tr><tr><td> HyperCyt vedhæftet fil </td><td> Intellicyt </td><td> NA </td><td></td></tr><tr><td> NS500</td><td> NanoSight </td><td> NA </td><td></td></tr></tbody></table>

evaluation of polymeric gene delivery nanoparticles by nanoparticle tracking analysis and high-throughput flow cytometry

Ikke-virale gen-levering ved anvendelse af polymere nanopartikler har vist sig som en attraktiv fremgangsmåde til genterapi til behandling af genetiske sygdomme 1 og som en teknologi til regenerativ medicin 2. Modsætning til vira, som har væsentlige sikkerhedsproblemer kan polymere nanopartikler være udformet til at være ikke-toksisk, ikke-immunogen, ikke-mutagene, lettere at syntetisere, kemisk alsidig, kan bære større nukleinsyre fragt og bionedbrydelige og / eller miljømæssigt responsiv. Kationiske polymerer selv samle med negativt ladet DNA via elektrostatisk interaktion til dannelse af komplekser af størrelsesordenen 100 nm, som normalt betegnes polymere nanopartikler. Eksempler på biomaterialer anvendes til dannelse nanoskala polykationiske genafgivelsessystemer nanopartikler indbefatter polylysin, polyphosphoesters, poly (amidoaminer) s og polyethylenimin (PEI), som er en ikke-nedbrydelige off-the-shelf kationiske polymer almindeligvis anvendes til nukleinsyreadministrationskompleks 1,3. Poly (beta-aminoester) s (PBAEs) er en nyere klasse af kationiske polymerer 4, som er hydrolytisk nedbrydelig 5,6 og har vist sig at være effektive til genafgivelse til svære at transficere celletyper, såsom humane retinale endotelceller (HRECs) 7, muse mamma-epiteliale celler 8, menneskelige hjerne cancerceller 9 og makrovaskulære (human navlevene, HUVEC&#39;er) endotelceller 10. En ny protokol til at karakterisere polymere nanopartikler anvender nanopartikler tracking analyse (NTA) er beskrevet. I denne fremgangsmåde er både partikelstørrelsesfordelingen og fordelingen af antallet af plasmider pr partikel opnået 11. Endvidere er et high-throughput 96-brønds plade transfektionsassay til hurtig screening af transfektion effektiviteten af ​​polymere nanopartikler præsenteret. I denne protokol, er poly (beta-amino ester) s (PBAEs) anvendes som model polymerer og humane retinale endotelceller (HRECs) anvendes som model humane celler. Denne protokol kan let tilpasses til at evaluere en polymer nanopartikel og enhver celletype af interesse i en multi-brønds pladeformat.

Figur 1 viser et fluorescensmikroskopi billede af et eksempel på en succesfuld transfektion af HRECs med EGFP plasmid. The brightfield billede nyttigt at sikre, at celler bevarer deres normale morfologi. Derudover kan cellelevedygtighedsassays, såsom MTS eller lignende assays, anvendes til at vurdere den nanopartikel toksicitet 7. Flowcytometri, som beskrevet, kan anvendes til at kvantificere transfektionseffektivitet. Ved brug af HyperCyt multi-brønds plade fastgørelse, bliver dataene sk...

Watch this Scientific Journal Video about Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry at JoVE.com

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

En protokol til nanopartikler sporing analyse (NTA) og højkapacitetsidentifikation flowcytometri for at evaluere polymere genafgivelsessystemer nanopartikler beskrives. NTA anvendes til at karakterisere nanopartikel partikelstørrelsesfordeling og plasmidet pr partikelfordeling. High-throughput flowcytometri muliggør kvantitativ transfektion Effektevalueringen for et bibliotek af gen-leveringsvehikler biomaterialer.

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