title: Development of a method to quantify the DNA content in cationic peptide-DNA nanoparticles.
abstract: Gene therapy has the potential to provide safe and targeted therapies for a variety of diseases. A range of intracellular gene delivery vehicles have been proposed for this purpose. Non-viral vectors are a particularly attractive option and among them cationic peptides have emerged as promising candidates. For the pharmaceutical formulation and application to clinical studies it is necessary to quantify the amount of pDNA condensed with the delivery system. There is a severe deficiency in this area, thus far no methods have been reported specifically for pDNA condensed with cationic peptide to form nanoparticles. The current study seeks to address this and describes the evaluation of a range of disruption agents to extract DNA from nanoparticles formed by condensation with cationic fusogenic peptides RALA and KALA. Only proteinase K exhibited efficient and reproducible results and compatibility with the PicoGreen reagent based quantification assay. Thus we report for the first time a simple and reliable method that can quantify the pDNA content in pDNA cationic peptide nanoparticles.
authors.affiliation: School of Pharmacy, Medical Biology Centre, Queen's University Belfast, 97 Lisburn Road, Belfast BT9 7BL, Northern Ireland, UK; Weatherall Institute of Molecular Medicine, MRC Molecular Haematology Unit, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DS, UK.
authors.forename: Arvind K
authors.initials: AK
authors.lastname: Jain
authors.affiliation: School of Pharmacy, Medical Biology Centre, Queen's University Belfast, 97 Lisburn Road, Belfast BT9 7BL, Northern Ireland, UK.
authors.forename: Helmy
authors.initials: H
authors.lastname: Yusuf
authors.affiliation: School of Pharmacy, Medical Biology Centre, Queen's University Belfast, 97 Lisburn Road, Belfast BT9 7BL, Northern Ireland, UK.
authors.forename: Aditya
authors.initials: A
authors.lastname: Pattani
authors.affiliation: School of Pharmacy, Medical Biology Centre, Queen's University Belfast, 97 Lisburn Road, Belfast BT9 7BL, Northern Ireland, UK.
authors.forename: Helen O
authors.initials: HO
authors.lastname: McCarthy
authors.affiliation: Centre for Vision and Vascular Science, Queen's University Belfast, Belfast, Northern Ireland, UK.
authors.forename: Denise M
authors.initials: DM
authors.lastname: McDonald
authors.affiliation: School of Pharmacy, Medical Biology Centre, Queen's University Belfast, 97 Lisburn Road, Belfast BT9 7BL, Northern Ireland, UK. Electronic address: v.kett@qub.ac.uk.
authors.forename: Vicky L
authors.initials: VL
authors.lastname: Kett
mesh_terms: D002412:Cations; D004247:DNA / Q000737:chemistry / Q000302:isolation & purification*; D004268:DNA-Binding Proteins / Q000737:chemistry*; D019286:Endopeptidase K / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D018014:Gene Transfer Techniques; D053758:Nanoparticles; D009690:Nucleic Acid Conformation; D009930:Organic Chemicals / Q000737:chemistry; D010316:Particle Size; D010455:Peptides / Q000737:chemistry*; D011487:Protein Conformation; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D013696:Temperature; D013997:Time Factors
chemical_list: D002412:Cations; D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; C105114:KALA amphipathic peptide; D009930:Organic Chemicals; D010455:Peptides; C099476:PicoGreen; C000596539:RALA peptide; D012967:Sodium Dodecyl Sulfate; D004247:DNA; D019286:Endopeptidase K
keywords: Cationic peptide; DNA quantification; KALA nanoparticles; PicoGreen; Proteinase K