Name: ex vivo red blood cell hemolysis assay for the evaluation of ph-responsive endosomolytic agents for cytosolic delivery of biomacromolecular drugs
Uploaded: 2013-03-09T14:00:00
Description: Watch this Scientific Journal Video about Ex Vivo Red Blood Cell Hemolysis Assay for the Evaluation of pH-responsive Endosomolytic Agents for Cytosolic Delivery of Biomacromolecular Drugs at JoVE.com

Forfatterne anerkender finansiering gennem Department of Defense Kongressen Instrueret Medical Research Programmer (# W81XWH-10-1-0445), National Institutes of Health (NIH R21 HL110056), og American Heart Association (# 11SDG4890030).

1. Forberedelse og Sterilisation af Buffere og testmidler <ol><li> 150 mM NaCI-puffer: Opløs 4,383 g NaCl krystaller i 500 ml nanorent vand. </li><li> pH buffer: Forbered phosphatpuffere ved pH 5,6, 6,2, 6,8 og 7,4 ved at blande passende mængder af monobasisk og dibasisk natriumphosphat. Hvis prøver skal testes ved lavere pH-værdier (dvs. pH &lt;5,6) derefter en mere hensigtsmæssig puffer, såsom citratbuffer, bør anvendes. Buffer opskrifter er let tilgængelige, og et eksempel Anmodningen er blevet til...

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pH-reagerende polymerer eller andre midler beregnet til endosomolytic funktion kan hurtigt og effektivt screenet baseret på lyse af røde blodlegemer ved pH-værdier forekommer i endosomet (figur 1; pH 6,8 - tidlige endosom, pH 6,2 - sene endosom, pH 5,6 - lysosom). 14-17 pH-afhængig hæmolyse er blevet anvendt til at screene luftfartsselskabernes muligheder for at mediere endosomale frigivelse af biomacromolecular terapeutika (f.eks peptider, siRNA, ODN&#39;er, proteiner), og resu...

Selvom der er mange potentielle slagfaste terapeutiske mål inde i cellen, den intracellulære afgivelse af midler er en betydelig udfordring. Ofte er stoffer, især biologiske stoffer, internaliseres af celler og solgt til vesikler, der enten føre til nedbrydning af indholdet gennem endo-lysosomal vej, eller shuttled back ud af cellen ved hjælp af eksocytose. 1. I den sidstnævnte proces, den interne pH af vesiklerne syrnes til omkring 5-6, som er det optimale pH for aktiviteten af enzymer, der fungerer i dette rum, såsom lysozym. 2 For nylig har en række materialer er specielt udviklet til at udnytte forsuring af endosomer at lette cytosolisk levering af deres last. Et eksempel på denne fremgangsmåde anvender syntetiske, polymere micelle nanopartikler, hvis kerne er zwitterioniske og charge-neutral ved fysiologisk pH (dvs. 7,4). Men ved pH 6,0- 6,5, polymererne bliver protoneret og opnå en positiv nettoladning, der destabiliserer micellen kerne, og de eksponerede polymersegmenter interagere med og forstyrre den endosomale membran. Denne aktivitet er blevet vist at fremme den endosomale udslip af peptid-og nucleinsyre-baserede terapeutiske midler, så de kan få adgang til deres cytosoliske mål. 3,4 Andre eksempler på metoder, der udvikles til at mediere endosomale escape at forstyrre membranbarrieren omfatter &quot;fusogene&quot; peptider eller proteiner, der kan mediere membranfusion eller forbigående poredannelse i phospholipid-dobbeltlag. fem Homopolymerer af anioniske alkyl-acryl-syrer, såsom poly (propylacrylic syre) er en anden velundersøgte tilgang, og i disse polymerer, dikterer protonering tilstand af vedhæng carboxylsyre overgangen i en hydrofob, membran-forstyrrende i endo-lysosomale pH-områder. 6,7 En nyttig model for screening endosomolytic opførsel er ex vivo pH-afhængig hæmolyse-assayet. 8. I dette modelsystem, at erythrocytmembranen tjener som et surrogat for lipiddobbeltlaget membran, der omslutter endo-lysosomale vesikler. Denne generalisere model er blevet anvendt af andre til at evaluere endosomolytic adfærd cellepenetrerende peptider og andre polymere genleveringssystemer. 8-11 I dette eksperiment blev humane røde blodlegemer og testmaterialer er co-inkuberet i buffere ved definerede pH-værdier, der efterligner ekstracellulære (7,4), tidlig endosomale (6,8), og sene endo-lysosomal (&lt;6,8) miljøer. Mængden af ​​hæmoglobin frigjort under inkubationsperioden kvantificeres som et mål for røde blodlegemer lysis, som normaliseres til mængden af ​​hæmoglobin frigjort i positive kontrolprøver lyseret med et detergent. Fra screening af et lille bibliotek af potentielt endosomolytic teststoffer, kan man udlede, at prøver, der producerer ingen hæmolyse ved pH 7,4, men signifikant forhøjet hemolysis ved pH &lt;6,5, vil være de mest effektive og cytocompatible kandidater til cytosolisk lægemiddelafgivelse. Materialer, som passer disse kriterier forventes at være inert og ikke vilkårligt ødelægge lipiddobbeltlagsmembraner (dvs. der kan forårsage cytotoksicitet) før de er blevet udsat for et fald i den lokale pH-værdi efter internalisering til endo-lysosomale rum. I denne protokol, er erythrocytter isoleret fra en menneskelig donor og co-inkuberet ved pH 5,6, 6,2, 6,8, eller 7,4 med eksperimentelle endosomolytic drug delivery agenter. Intakte erythrocytter pelleteres, og supernatanterne (som indeholder hæmoglobin frigivet fra lyserede erythrocytter) analyseres for den karakteristiske absorbans af hæmoglobin via en pladelæser (figur 1).

<table border="1"><tr><td> Navn på reagenset </td><td> Firma </td><td> Katalognummer </td><td> Kommentarer (valgfrit) </td></tr><tr><td> BD Vacutainer - K2EDTA Vacutainer-rør </td><td> Fisher Scientific </td><td> 22-253-145 </td><td> Til blodprøvetagning </td></tr><tr><td> BD Vacutainer Blodtapningsprocedurerne Needles, 20,5-gauge </td><td> Fisher Scientific </td><td> 02-665-31 </td><td> Til blodprøvetagning </td></tr><tr><td> BD Vacutainer Tube Holder / Needle Adapter </td><td> Fisher Scientific </td><td> 22-289-953 </td><td> Til blodprøvetagning </td></tr><tr><td> BD Brand Isopropyl Alcohol Swabs </td><td> Fisher Scientific </td><td> 13-680-63 </td><td> Til blodprøvetagning </td></tr><tr><td> BD Vacutainer Latex-Free Tourniquet </td><td> Fisher Scientific </td><td> 02-657-6 </td><td> Til blodprøvetagning </td&gt; </tr><tr><td> Saltsyre (konc.) </td><td> Fisher Scientific </td><td> A144-500 </td><td> Til justering af pH i D-PBS. </td></tr><tr><td> Triton X-100 </td><td> Sigma-Aldrich </td><td> T8787 </td><td> Positiv kontrol </td></tr><tr><td> Dulbeccos PBS </td><td> Invitrogen </td><td> 14.190 </td><td></td></tr><tr><td> Nalgene MF75 Steril engangsflaske-Top filterenhed med SFCA Membrane </td><td> Fisher Scientific </td><td> 09 til 740-44A </td><td></td></tr><tr><td> BD 96-brønds plader, fladbundet, vævskultur-behandlet polystyren </td><td> Fisher Scientific </td><td> 08 til 772-2C </td><td> For plade-læser ved afslutningen af ​​assayet. </td></tr><tr><td> BD 96-brønds plader, rundbundet, vævskultur-behandlet polystyren </td><td> Fisher Scientific </td><td> 08-772-17 </td><td> Til inkubation af røde blodlegemer med eksperimentelle midler. </td></tr></table&gt;

ex vivo red blood cell hemolysis assay for the evaluation of ph-responsive endosomolytic agents for cytosolic delivery of biomacromolecular drugs

Phospholipid-dobbeltlag, der udgør endo-lysosomale vesikler kan være til hinder for levering af biologiske lægemidler til intracellulære mål. For at overvinde denne barriere, er et antal syntetiske lægemiddelbærere blevet manipuleret til aktivt forstyrre den endosomale membran og levere lasten til cytoplasmaet. Her beskriver vi hæmolyse-assayet, der kan anvendes som en hurtig, high-throughput screening for cytocompatibility og endosomolytic aktivitet af intracellulære lægemiddeladministrationssystemer. I hæmolyse assay humane røde blodlegemer og testmaterialer co-inkuberes i buffere ved definerede pH-værdier, der efterligner ekstracellulære, tidlige endosomale, og sene endo-lysosomale miljø. Efter et centrifugeringstrin for at pelletere intakte røde blodlegemer, er mængden af ​​hæmoglobin frigjort i mediet spektrofotometrisk måles (405 nm for bedste dynamisk område). Den procentvise røde blodlegemer forstyrrelser derefter kvantificeret i forhold til positiv kontrol Samples lyseret med et rengøringsmiddel. I dette modelsystem erythrocytmembranen tjener som et surrogat for lipiddobbeltlaget membran, der omslutter endo-lysosomale vesikler. Det ønskede resultat er ubetydelig hemolyse ved fysiologisk pH (7,4) og robust hæmolyse i endo-lysosomal pH-område fra ca pH 5-6,8.

<html> Typisk er midler, der udviser ideelle pH-afhængig hæmolytisk adfærd har det største potentiale for cytosolisk afgivelse af lægemidler, nukleinsyrer eller andre bioaktive molekyler. Dette eksemplificeres ved Agent # 1 som gengivet i figur 2, der udviser minimal hæmolyse ved pH 7,4, men en drastisk stigning i hæmolytisk adfærd ved endosomale pH-områder (&lt;6.5). Nogle midler kan udvise en høj grad af hæmolytisk adfærd ved fysiologiske pH-områder (befuldmægtiget # 2 ved 40 ug / ml, <st...

Watch this Scientific Journal Video about Ex Vivo Red Blood Cell Hemolysis Assay for the Evaluation of pH-responsive Endosomolytic Agents for Cytosolic Delivery of Biomacromolecular Drugs at JoVE.com

Ex Vivo Red Blood Cell Hemolysis Assay for the Evaluation of pH-responsive Endosomolytic Agents for Cytosolic Delivery of Biomacromolecular Drugs

En hæmolyse-assayet kan anvendes som en hurtig, high-throughput screen for lægemiddeltilførselssystemer &#39;cytocompatibility og endosomolytic aktivitet for intracellulær last levering. Assayet måler afbrydelsen af ​​erythrocytmembraner som funktion af miljømæssige pH.

Ex Vivo Red Blood Cell Hæmolyse Assay for Vurdering af pH-responsive Endosomolytic Agents for cytosolisk Levering af Biomacromolecular Drugs

Phospholipid bilayers that constitute endo-lysosomal vesicles can pose a barrier to delivery of biologic drugs to intracellular targets. To overcome this ...

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Ex Vivo Red Blood Cell Hemolysis Assay for the Evaluation of pH-responsive Endosomolytic Agents for Cytosolic Delivery of Biomacromolecular Drugs

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