Biomedical subjects

Brian A Lobo

Publications and source records attributed to Brian A Lobo.

5 recordsLinked to original sources

PubMed2003

Thermodynamic analysis of binding and protonation in DOTAP/DOPE (1:1): DNA complexes using isothermal titration calorimetry.

A better understanding of the nature of the interaction between various cationic lipids used for gene delivery and DNA would lend insight into their structural and physical properties that may modulate their efficacy. We therefore separated the protonation and binding events which occur upon complexation of 1:1 DOTAP (1,2-dioleoyl-3-trimethylammonium propane):DOPE (1,2-dioleoylphosphatidylethanolamine) liposomes to DNA using proton linkage theory and isothermal titration calorimetry (ITC). The enthalpy of DOPE protonation was estimated as -45.0+/-0.7 kJ/mol and the intrinsic binding enthalpy of lipid to DNA as +2.8+/-0.3 kJ/mol. The pK(a) of DOPE was calculated to shift from 7.7+/-0.1 in the free state to 8.8+/-0.1 in the complex. At physiological ionic strength, proton linkage was not observed upon complex formation and the buffer-independent binding enthalpy was +1.0+/-0.4 kJ/mol. These studies indicate that the intrinsic interaction between 1:1 DOTAP/DOPE and DNA is an entropy-driven process and that the affinities of cationic lipids that are formulated with and without DOPE for DNA are controlled by the positive entropic changes that occur upon complex formation.

Binding Sites↗

PubMed2003

Biophysical characterization of PEI/DNA complexes.

The main goal of this study was to determine the effects of polyethylenimine (PEI) molecular weight and structure (750 kDa, 25 kDa, 2 kDa branched, and 25 kDa linear PEI) and the nitrogen/phosphate (N/P) molar ratio on the physical properties and transfection efficiencies of PEI/DNA complexes. Fourier transform infrared spectroscopy revealed that DNA remained in the B conformation when complexed to all PEIs. Unique alterations in the circular dichroism spectra of DNA were observed in the presence of each PEI, whereas differential scanning calorimetry measurements showed that all PEIs examined destabilized supercoiled DNA at N/P < 3/1, but not at higher ratios. Isothermal titration calorimetry revealed the existence of protonation changes at low ionic strength due to possible shifts in pK(a) of the ionizable groups of PEI during complex formation. Twenty-five kilodalton branched and 25 kDa linear PEI complexes showed the highest transfection efficiencies at an N/P ratio of 6:1 in COS-7 and CHO-K1 cells, respectively. These investigations have detected alterations in the physical and colloidal properties of the complexes that were sensitive to polymer structure, molecular weight, and polymer/DNA ratio, but these properties did not directly correlate with their transfection efficiencies. To further probe any possible relationship between these parameters and activity, a more refined biophysical analysis of any subpopulations in these samples that may differ in transfection activity is suggested, although the existence of such species remains unknown.

Animals↗

PubMed2003

Structure/function analysis of peptoid/lipitoid:DNA complexes.

Previous transfection studies of cationic peptoid polymers (N-substituted polyglycines) and cationic lipitoid polymers (peptoid-phospholipid conjugates) have shown that only the polymers which possessed a repeating (cationic, hydrophobic, hydrophobic) substituent sequence are efficient in gene transfer in vitro. To determine if there is a physical attribute of peptoid and lipitoid complexes that correlates with efficient gene transfection, biophysical, and transfection measurements were performed with polymer:DNA complexes containing each of seven structurally diverse peptoid polymers and two lipitoids that possess different hydrophobic substituents. These measurements revealed that the biophysical properties of these complexes (size, zeta-potential, ethidium bromide exclusion) varied with polymer structure and complex (+/-) charge ratio but were not directly predictive of transfection efficiency. Unique alterations in the circular dichroism spectra of DNA were observed in complexes containing several of the peptoids and both lipitoids, although FTIR spectroscopy demonstrated that the DNA remained in the B-form. The lack of correlations between the physical properties and the transfection activities of these polyplexes suggests that a further subpopulation examination of these complexes by these methods may reveal hidden structure-activity relationships.

Animals↗

PubMed2002

Differential scanning calorimetric studies of the thermal stability of plasmid DNA complexed with cationic lipids and polymers.

The thermal stabilities of supercoiled (SC) and linear/open circular (LIN/OC) forms of plasmid DNA when complexed with cationic lipids or cationic polymers used for cellular transfection were assessed using differential scanning calorimetry. Differences in the stability of SC DNA produced by the cationic lipids DOTAP (1,2-dioleoyltrimethyl ammoniumpropane chloride), DSTAP (1,2-distearyltrimethyl ammoniumpropane chloride), and DDAB (dimethyldioctadecylammonium bromide) upon complexation suggest possible effects of headgroup structure on the stability of SC DNA and minimal effects of lipid acyl chain saturation/unsaturation. Complexation of DNA with the cationic polymers polyethylenimine (PEI) or poly-L-lysine (PLL) (but not poly-L-arginine) resulted in a decreased stability of SC DNA when the DNA was in charge excess, although all polymers stabilized SC DNA when the polymer was in charge excess. The effects of these cationic polymers on the stability of SC DNA can be explained by changes produced in the tertiary structure of SC DNA upon binding and may reflect the importance of the topological constraint of supercoiling upon the stability of the resulting complexes.

Calorimetry, Differential Scanning↗

PubMed2002

Enhancements in gene expression by the choice of plasmid DNA formulations containing neutral polymeric excipients.

Formulations containing maltodextrin (2% w/v) were identified to facilitate intramuscular (im) delivery of plasmid DNA in mice using the reporter genes luciferase and chloramphenicol acetyltransferase (CAT) and the therapeutic gene of erythropoietin (EPO) as monitors of transfection efficiency. Even though considerable variability in gene expression was observed in animals, a 5-8-fold enhancement of reporter gene expression was observed with this excipient compared with saline formulations of DNA. In a therapeutically significant experiment, a single im injection of an EPO plasmid formulation containing 2% (w/v) maltodextrin resulted in a significant and prolonged elevation of the hematocrit levels of mice compared with control DNA in saline. Biophysical studies with Fourier transform infrared (FTIR) spectroscopy, isothermal titration, and differential scanning calorimetry (DSC) suggested a weak interaction between DNA and maltodextrin as well as a thermal stabilizing effect on the DNA. These in vivo and biophysical results with maltodextrin are comparable to those reported previously with other nonionic polymers, such as poly(vinyl pyrrolidone) and poloxamers, and indicate that maltodextrin is an additional nonionic excipient that displays the property of gene expression enhancement.

Animals↗