Delivery of polyethylenimine/DNA complexes assembled in a microfluidics device.
Polyethylenimine (PEI) and plasmid DNA (pDNA) complexes (PEI/pDNA) are nonviral vectors for gene delivery. The conventional method for producing these complexes involves bulk mixing (BM) of PEI and DNA followed by vortexing which at low N/P ratios results in large particle size distribution, low cytotoxicity, and poor gene transfection, while at high N/P ratios it results in small particle size and better gene transfection but high cytotoxicity. To improve size control, gene transfection efficiency, and cytotoxicity, in this study, we used a microfluidic hydrodynamic focusing (MF) device to prepare PEI/pDNA complexes at N/P = 3.3 and 6.7. We used bulk mixing as control, mouse NIH 3T3 fibroblast cells and mouse embryonic stem (mES) cells as model cell lines, plasmid encoding green fluorescent protein (pGFP) and secreted alkaline phosphatase (pSEAP) as the reporter gene, and commercially available Lipofectamine 2,000 as a positive control. The complexes were characterized by atomic force microscopy (AFM), dynamic light scattering (DLS), and zeta potential (zeta) measurement. Confocal laser scanning microscopy (CLSM) and fluorescent labeling techniques were used to visualize the complex size distribution, complexation uniformity, and cellular distribution. The results showed that MF produced complexes were smaller and more uniformly complexed and had higher cell viability and improved exogenous gene expression.
Koh
Chee Guan
CG
Department of Chemical and Biomolecular Engineering, The Ohio State University, 140 West 19th Avenue, Columbus, Ohio 43210, USA.
Kang
Xihai
X
Xie
Yubing
Y
Fei
Zhengzheng
Z
Guan
Jingjiao
J
Yu
Bo
B
Zhang
Xulang
X
Lee
L James
LJ
D000469:Alkaline Phosphatase / Q000235:genetics; D000818:Animals; D002470:Cell Survival; D002478:Cells, Cultured; D004274:DNA, Recombinant / Q000008:administration & dosage* / Q000235:genetics; D016503:Drug Delivery Systems; D005456:Fluorescent Dyes; D015870:Gene Expression; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D051379:Mice; D046210:Microfluidic Analytical Techniques; D018625:Microscopy, Atomic Force; D018613:Microscopy, Confocal; D041681:NIH 3T3 Cells; D053758:Nanoparticles / Q000737:chemistry; D036103:Nanotechnology; D010957:Plasmids / Q000008:administration & dosage / Q000235:genetics; D011094:Polyethyleneimine / Q000737:chemistry*; D012542:Scattering, Radiation; D014162:Transfection
D004274:DNA, Recombinant; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins; D011094:Polyethyleneimine; D000469:Alkaline Phosphatase