Multilayered thin films from poly(amido amine)s and DNA.
Dip-coated multilayered thin films of poly(amido amine)s (PAAs) and DNA have been developed to provide surfaces with cell-transfecting capabilities. Three types of PAAs, differing in side chain functional groups, were synthesized and characterized for their properties in forming multilayered structures with ultrasonicated calf thymus DNA (CTDNA) as model DNA. All three polymers display a multilayer build-up in linear profiles as demonstrated by UV spectroscopy. More highly charged side chains were found to provide the lowest deposition of DNA. Surface profiles of the obtained films were investigated by atomic force microscopy (AFM) and static water contact angle measurements to reveal complete surface coverage after at least four layer pair depositions, where alternating patterns of surface profiles were observed depending on whether the cationic polymer or the anionic DNA layer was on top. The stability of the formed surfaces was investigated in vitro under physiological and reductive conditions. Owing to the presence of disulfide bonds in the PAA main chain, the films were readily degraded in the presence of 1mM of DTT in vitro. Under non-reductive physiological conditions, two of the thicker films underwent thermodynamic rearrangement, which resulted in release of approximately half of the incorporated material within 1h, which was caused by the physiological salt concentration. Further, this unpacking phenomenon proved useful in transfecting COS-7 cells seeded on top of these multilayers containing functional plasmid DNA encoding for green fluorescence protein (GFP). Two out of the three different multilayers facilitated good COS-7 cell attachment, proliferation, and transfection in vitro within 2d ays of culture. Fluorescence staining further revealed the presence of DNA-containing released film material among cultured cells. The present work demonstrates the possibility of coating surfaces with thin films that are conveniently adjustable in thickness and amount of active agent to provide cell-transfecting functionality. In this manner transfection can be achieved by simply culturing cells on a multilayer-coated surface in their optimal culture condition (in the presence of serum) and without the need of removing the transfection agent to avoid cytotoxicity.
Department of Controlled Drug Delivery, MIRA Institute for Biomedical Technology and Technical Medicine, Faculty of Science and Technology, University of Twente, P.O. Box 217, 7500 AE Enschede, The Netherlands.
Sry D
SD
Hujaya
Department of Controlled Drug Delivery, MIRA Institute for Biomedical Technology and Technical Medicine, Faculty of Science and Technology, University of Twente, P.O. Box 217, 7500 AE Enschede, The Netherlands. Electronic address: J.F.J.Engbersen@utwente.nl.
Johan F J
JF
Engbersen
Department of Controlled Drug Delivery, MIRA Institute for Biomedical Technology and Technical Medicine, Faculty of Science and Technology, University of Twente, P.O. Box 217, 7500 AE Enschede, The Netherlands. Electronic address: J.M.J.Paulusse@utwente.nl.
Jos M J
JM
Paulusse
D000818:Animals; D019556:COS Cells; D002417:Cattle; D002470:Cell Survival; D002522:Chlorocebus aethiops; D002850:Chromatography, Gel; D004247:DNA / Q000737:chemistry*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008970:Molecular Weight; D011073:Polyamines / Q000138:chemical synthesis / Q000737:chemistry*; D066244:Proton Magnetic Resonance Spectroscopy; D014162:Transfection; D014867:Water / Q000737:chemistry; D017155:Wettability
C531249:Poly(amidoamine); D011073:Polyamines; D014867:Water; D049452:Green Fluorescent Proteins; D004247:DNA; C427203:calf thymus DNA
Biodegradable polymers; Gene delivery; Layer-by-layer assembly; Multilayered thin films; Transfection