Nano-carriers for DNA delivery to the lung based upon a TAT-derived peptide covalently coupled to PEG-PEI.
Gene therapy aimed at the respiratory epithelium holds therapeutic potential for diseases such as cystic fibrosis and lung cancer. Polyethylenimine (PEI) has been utilized for gene delivery to the airways. In this study, we describe a new modification of PEI, in which an oligopeptide related to the protein transduction domain of HIV-1 TAT was covalently coupled to 25 kDa PEI (PEI) through a heterobifunctional polyethylenglycol (PEG) spacer resulting in a TAT-PEG-PEI conjugate. Improved DNA reporter gene complexation and protection was observed for small (approximately 90 nm) polyplexes as well as significantly improved stability against polyanions, Alveofact, bronchial alveolar lining fluid and DNase. To determine polyplex toxicity in vitro, MTT assays were performed and, for in vivo testing, the mice bronchial alveolar lavage was investigated for total cell counts, quantity of neutrophils, total protein and TNF-alpha concentration. All parameters suggest significantly lower toxicity for TAT-PEG-PEI. Transfection efficiencies of both PEI and TAT-PEG-PEI polyplexes with DNA were studied under in vitro conditions (A549) and in mice after intratracheal instillation. While luciferase expression in A549 cells was much lower for TAT-PEG-PEI (0.2 ng/mg protein) than for PEI (2 ng/mg), significantly higher transfection efficiencies for TAT-PEG-PEI were detected in mice. Reporter gene expression was distributed through bronchial and alveolar tissue. Thus, TAT-PEG-PEI represents a new approach to non-viral gene carriers for lung therapy, comprising protection for plasmid DNA, low toxicity and significantly enhanced transfection efficiency under in vivo conditions.
Kleemann
E
E
Department of Pharmaceutics and Biopharmacy, Philipps-University, Marburg, Germany.
Neu
M
M
Jekel
N
N
Fink
L
L
Schmehl
T
T
Gessler
T
T
Seeger
W
W
Kissel
T
T
D000818:Animals; D001992:Bronchoalveolar Lavage Fluid / Q000737:chemistry; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D004247:DNA / Q000008:administration & dosage*; D004337:Drug Carriers; D016503:Drug Delivery Systems; D004847:Epithelial Cells / Q000502:physiology; D004996:Ethidium / Q000737:chemistry; D015696:Gene Products, tat / Q000737:chemistry* / Q000633:toxicity; D017930:Genes, Reporter / Q000235:genetics; D015316:Genetic Therapy / Q000379:methods*; D006493:Heparin / Q000737:chemistry; D006801:Humans; D008156:Luciferases / Q000235:genetics; D008168:Lung / Q000166:cytology / Q000473:pathology; D051379:Mice; D049329:Nanostructures; D023282:Nuclease Protection Assays; D009841:Oligonucleotides / Q000008:administration & dosage; D011092:Polyethylene Glycols / Q000737:chemistry*; D011094:Polyethyleneimine / Q000737:chemistry*; D014018:Tissue Distribution; D014162:Transfection
D004337:Drug Carriers; D015696:Gene Products, tat; D009841:Oligonucleotides; D011092:Polyethylene Glycols; D011094:Polyethyleneimine; D006493:Heparin; D004247:DNA; D008156:Luciferases; D004996:Ethidium