title: Thermodynamic aspects and biological profile of CDAN/DOPE and DC-Chol/DOPE lipoplexes.
abstract: The DNA complexation and condensation properties of two established cationic liposome formulations, CDAN/DOPE (50:50, m/m; Trojene) and DC-Chol/DOPE (60:40, m/m), were investigated by using a combination of isothermal titration calorimetry (ITC), circular dichroism (CD), photon correlation spectroscopy (PCS), and turbidity assays. Plasmid DNA (7528 bp) was titrated with extruded liposomes (90 +/- 15 nm) and a thermodynamic profile established. ITC data revealed that the two liposome formulations differ substantially in their DNA complexation characteristics. Equilibrium dissociation constants for CDAN/DOPE (K(d) = 19 +/- 3 microM) and DC-Chol/DOPE liposomes (K(d) = 2 +/- 0.5 microM) were obtained by fitting the experimental data in a one-site binding model. Both CDAN/DOPE and DC-Chol/DOPE binding events take place with a negative binding enthalpy (DeltaH degrees = -0.5 and -1.7 kcal/mol, respectively) and increasing system entropy (TDeltaS = 6 +/- 0.3 and 6.2 +/- 0.3 kcal/mol, respectively). Interestingly, CDAN/DOPE liposomes undergo substantial rehydration and protonation prior to complexation with pDNA, which is observed as two discrete exothermic signals during titration. No such biphasic effects are seen with respect to the binding between DC-Chol/DOPE and pDNA that appears to be otherwise instantaneous with no rehydration effects. The rehydration and protonation characteristics of CDAN/DOPE liposomes in comparison with those of DC-Chol/DOPE cationic liposomes are confirmed by ITC; CDAN/DOPE liposomes have strongly exothermic dilution characteristics and DC-Chol/DOPE liposomes only mildly endothermic characteristics. Furthermore, analysis of cationic liposome-pDNA binding by CD spectroscopy reveals that CDAN/DOPE-pDNA lipoplexes are more structurally fluid than DC-Chol/DOPE-pDNA lipoplexes. CDAN/DOPE liposomes induced considerable fluctuation in the DNA structure for at least 60 min, whereas liposomes obtained from DC-Chol/DOPE lack the same effect on the DNA structure. Turbidity studies show that DC-Chol/DOPE lipoplexes exhibit greater resistance to serum than CDAN/DOPE lipoplexes, which showed substantial precipitation after incubation for 100 min with serum. Transfection studies on HeLa and Panc-1 cells reveal that CDAN/DOPE lipoplexes are superior in efficacy to DC-Chol/DOPE lipoplexes. CDAN/DOPE liposomes tend to transfect best in normal growth medium (including 10% serum and antibiotics), whereas DC-Chol/DOPE lipoplexes transfect best under serum free transfection conditions.
authors.affiliation: IC-Vec Ltd., Flowers Building, Armstrong Road, London SW7 2AZ, United Kingdom. m.keller@icvec.com
authors.forename: Michael
authors.initials: M
authors.lastname: Keller
authors.forename: Michael R
authors.initials: MR
authors.lastname: Jorgensen
authors.forename: Eric
authors.initials: E
authors.lastname: Perouzel
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Miller
mesh_terms: D055592:Biophysical Phenomena; D001703:Biophysics; D002151:Calorimetry; D002784:Cholesterol / Q000031:analogs & derivatives* / Q000737:chemistry*; D002942:Circular Dichroism; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D008081:Liposomes / Q000737:chemistry*; D009391:Nephelometry and Turbidimetry; D019906:Nuclear Magnetic Resonance, Biomolecular; D010714:Phosphatidylethanolamines / Q000737:chemistry*; D010957:Plasmids / Q000737:chemistry / Q000235:genetics; D013057:Spectrum Analysis; D013816:Thermodynamics; D014162:Transfection / Q000379:methods
chemical_list: C410262:1,2-dioleoyl-glycero-3-phosphatidyl ethanolamine; D008081:Liposomes; D010714:Phosphatidylethanolamines; C070648:3-(N-(N',N'-dimethylaminoethane)carbamoyl)cholesterol; D002784:Cholesterol