title: Multilayer mediated forward and patterned siRNA transfection using linear-PEI at extended N/P ratios.
abstract: Gene delivery from a substrate depends, in part, on the vector-nucleic acid complex that is bound to the surface and the cell adhesive properties of the surface. Here, we present a method to deliver patterns of small interfering RNA (siRNA) that capitalize on a forward transfection method (transfection by introducing siRNA transfection reagent complexes onto plated cells); herein denoted as multilayer mediated forward transfection (MFT). This method separates the substrate-mediated delivery from the cell adhesive properties of the surface. pH responsive layer-by-layer (LbL) assembled multilayers were used as the delivery platform and microcontact printing technique (microCP) was used to pattern nanoparticles of transfection reagent-siRNA complexes onto degradable multilayers. Efficient MFT depend on optimal formulation of the nanoparticles. 25 kDa linear polyethylenimine (LPEI) was optimized as the siRNA transfection reagent for normal forward transfection (NFT) of the nanoparticles. A broad range of LPEI-siRNA nitrogen/phosphate (N/P) ratios (ranging from 5 to 90) was evaluated for the relative amounts of siRNA incorporated into the nanoparticles, nanoparticle size and NFT efficiencies. All the siRNA was incorporated into the nanoparticles at N/P ratio near 90. Increasing the amount of siRNA incorporated into the nanoparticles, with increasing N/P ratio correlated with a linear blue shift in the ultraviolet/visible (UV/vis) absorbance spectrum of the LPEI-siRNA nanoparticles. NFT efficiency greater than 80% was achieved with minimal cytotoxicity at N/P ratio of 30 and siRNA concentration of 200 nM. Similarly, MFT efficiency 60% was achieved for LPEI-siRNA nanoparticles at N/P ratios greater than 30.
authors.affiliation: Department of Chemical Engineering and Materials Science, Michigan State University, East Lansing, MI 48824, USA.
authors.forename: Sumit
authors.initials: S
authors.lastname: Mehrotra
authors.forename: Ilsoon
authors.initials: I
authors.lastname: Lee
authors.forename: Christina
authors.initials: C
authors.lastname: Chan
mesh_terms: D016923:Cell Death; D004587:Electrophoresis, Agar Gel; D055785:Gene Knockdown Techniques; D006367:HeLa Cells; D006801:Humans; D008051:Lipid Bilayers; D008856:Microscopy, Fluorescence; D053758:Nanoparticles / Q000737:chemistry; D009584:Nitrogen / Q000737:chemistry*; D010316:Particle Size; D010710:Phosphates / Q000737:chemistry*; D011094:Polyethyleneimine / Q000737:chemistry*; D034741:RNA, Small Interfering / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013056:Spectrophotometry, Ultraviolet; D014162:Transfection / Q000379:methods*; D019892:eIF-2 Kinase / Q000235:genetics / Q000378:metabolism
chemical_list: D008051:Lipid Bilayers; D010710:Phosphates; D034741:RNA, Small Interfering; D011094:Polyethyleneimine; D019892:eIF-2 Kinase; D009584:Nitrogen