Alexa fluor 488 labeled sirna

PEI-PCL-PEG and PEI-PCL-PEG-FA copolymers were synthesized as described before.(22 (link)) Briefly, ring opening polymerization of polycaprolactone (PCL) and hetero-bifunctional (HO-PEG-COOH, 3.5 and 5 kDa) PEG was performed for the targeted polymer. For non-targeted PEI-PCL-PEG, monofunctional (CH3-PEG-COOH, 5 kDa) PEG (JenKem Technologies, United States) was used instead. Acrylate-PCL-b-PEG-alkyne or acrylate-PCL-b-mPEG was reacted with hyper branched polyethylenimine (hyPEI, 25k Da, BASF, Ludwigshafen, Germany) in a Michael addition, and azido functionalized folic acid was coupled to the alkyne-modified PEG in a click reaction. Firefly luciferase (FLuc) dicer substrate double-stranded siRNA (sense: 5′-pGGUUCCUGGAACAAUUGCUUUUAca-3′, antisense: 3′-GACCAAGGACCUUGUUAACGAAAAUGU-5′, where p denotes a phosphate residue, lower case bold letters are 2′-deoxyribonucleotides, capital letters are ribonucleotides and underlined capital letters are 2′-O-methylribonucleotides) (DsiRNA), and Alexa Fluor-488 labeled siRNA was purchased from Integrated DNA Technologies (IDT, Coralville, IA).

Hetero-bifunctional poly(ethylene glycol) (3.5 kDa), as well as methyl terminated monofunctional poly(ethylene glycol) (5 kDa) was purchased from JenKem Technologies (Plano, TX, USA) and chemically modified based on previously published protocols.[5 (link)] Hyperbranched polyethylenimine (hy-PEI, 25 kDa) was purchased from BASF (Ludwigshafen, Germany). Dicer substrate double-stranded siRNA (DsiRNA) targeting the Enhanced Green Fluorescent Protein gene (EGFP siRNA, 25/27), Firefly Luciferase (luc), and a Negative Control (scr), as well as Alexa Fluor-488 labeled siRNA were purchased from Integrated DNA Technologies (IDT, Coralville, IA, USA).[42 (link)] Folic acid depleted Dulbecco’s Modified Eagle’s Medium (10×) for cell culture, phosphate buffered saline (PBS), heat-inactivated fetal bovine serum (FBS), D-(+)-glucose, and sodium bicarbonate was bought from Sigma-Aldrich (St. Louis, MO, USA). The chelator pBn-SCN-Bn-DTPA was purchased from Macrocyclics (Plano, TX, USA) while arsenazo(III) was purchased from Chem-Impex INT’L INC (Wood Dale, IL, USA), and yttrium(III) chloride was obtained from Acros Organics (Geel, Belgium).