Molecular shape of the cationic lipid controls the structure of cationic lipid/dioleylphosphatidylethanolamine-DNA complexes and the efficiency of gene delivery.

Pyridinium amphiphiles, abbreviated as SAINT, are highly efficient vectors for delivery of DNA into cells. Within a group of structurally related compounds that differ in transfection capacity, we have investigated the role of the shape and structure of the pyridinium molecule on the stability of bilayers formed from a given SAINT and dioleoylphosphatidylethanolamine (DOPE) and on the polymorphism of SAINT/DOPE-DNA complexes. Using electron microscopy and small angle x-ray scattering, a relationship was established between the structure, stability, and morphology of the lipoplexes and their transfection efficiency. The structure with the lowest ratio of the cross-sectional area occupied by polar over hydrophobic domains (SAINT-2) formed the most unstable bilayers when mixed with DOPE and tended to convert into the hexagonal structure. In SAINT-2-containing lipoplexes, a hexagonal topology was apparent, provided that DOPE was present and complex assembly occurred in 150 mm NaCl. If not, a lamellar phase was obtained, as for lipoplexes prepared from geometrically more balanced SAINT structures. The hexagonal topology strongly promotes transfection efficiency, whereas a strongly reduced activity is seen for complexes displaying the lamellar topology. We conclude that in the DOPE-containing complexes the molecular shape and the nonbilayer preferences of the cationic lipid control the topology of the lipoplex and thereby the transfection efficiency.

Effect of purification followed by solubilization of receptor material on quantitative receptor assays for anticholinergic drugs.

In order to optimize quantitative receptor assays for anticholinergics, the different receptor preparations resulting from the purification and the solubilization of the P2 pellet from the calf striatum were evaluated. The dissociation constants for two chemically different anticholinergics, the tertiary amine scopolamine and the quaternary amine oxyphenonium, were calculated from inhibition studies of 3H-NMS binding in buffer and plasma. The Kd values for both anticholinergics were similar for all the membrane-bound receptor preparations (unpurified and the purified P2 pellet) either in buffer or in plasma. More pronounced differences were observed between the membrane-bound and solubilized receptors. By introducing the solubilized receptor as well, differences between the individual anticholinergics appeared. On the one hand, for scopolamine, a gain in sensitivity of 1.5-2.8 in plasma was observed for the solubilized receptor. On the other hand, in the case of oxyphenonium, a dramatic loss in sensitivity (by a factor of about 24) was observed with the solubilized receptor, as compared to the membrane-bound receptor, in buffer. Very interestingly, however, when the solubilized receptor was used in plasma, a lowering of the Kd value was found for both anticholinergics, i.e. the assays became more sensitive. Such an effect (not observed for the membrane-bound receptor) could be obtained only when the percentage of digitonin present in the assay was at least 0.12% (w/v) or higher.

Evaluation of a purification procedure for the muscarinic receptor for the purpose of quantitative receptor assays of anticholinergics. Part A: The membrane-bound receptor.

The presented purification procedure for the muscarinic receptor from calf striatum includes the extraction of lipids with hexane in the first step and the removal of 39% of non-receptor proteins with 2 M NaCl in the second step. The simplicity of such an approach to the purification of the receptor warrants its use in the routine practice for quantitative purposes. The high affinity binding of tertiary 3H-dexetimide (3H-DEX) and quaternary 3H-N-methylscopolamine (3H-NMS) is preserved after the removal of irrelevant lipids and proteins from the P2-pellet. The overall yield of receptors--80%, when labelled with 3H-NMS, was satisfactory. Moreover, the final product, the NaCl-pellet, exerts a higher density of 3H-NMS binding sites per mg proteins by a factor of about 1.7. The overall yield of receptors and purification factor were lower, when measured with 3H-DEX. The total yield of 3H-DEX binding sites amounted to about 40% and the receptor density per mg protein decreased by a factor of 0.85. We did not succeed in the improvement of the ratio specific/non-specific binding, neither for 3H-DEX nor for 3H-NMS for the purified receptor preparations. The use of 3H-NMS is preferable to 3H-DEX in plasma sample assays because of a negligible effect of plasma on ligand binding when compared with 3H-DEX.

Evaluation of a purification procedure for the muscarinic receptor for the purpose of quantitative receptor assays of anticholinergics. Part B: The solubilized receptor.

For the purpose of quantitative receptor assays, a three-step solubilization procedure including three optimization sets for muscarinic receptor from calf striatum was developed. The first step includes the extraction of the P2-pellet with n-hexane and consequently with 2 M NaCl. By the latter, 39% of non-receptor proteins was extracted. The resulting pellet (NaCl-pellet), enriched in muscarinic receptors by a factor of 1.5-1.7, was solubilized with 1% digitonin. The binding parameters of the solubilized receptor were determined for the tertiary 3H-dexetimide (3H-DEX) and the quaternary 3H-N-methylscopolamine (3H-NMS). The resulting receptor density measured with 3H-dexetimide was lower (43.3% of that for the NaCl-pellet) than that for 3H-N-methyl-scopolamine (56.7%). The treatment with digitonin preserved the high affinity for 3H-N-methylscopolamine (Kd = 0.645 nM), however the affinity of 3H-dexetimide decreased after solubilization (Kd = 8.526 nM). The use of solubilized receptors in combination with hydrophilic 3H-NMS allows to increase the ratio specific/non-specific binding, since the non-specific binding for this ligand to the solubilized preparation is lower when compared with membrane-bound receptors. The above solubilization procedure was found preferable over directly solubilizing the P2-pellet since (a) the receptor density for 3H-NMS was higher for the solubilized NaCl-pellet by a factor of about 1.7, and (b) the treatment of the P2-pellet with digitonin resulted in a lowering of the Kd to 2.422 nM. However, with respect to the plasma effect on the ligand binding, both solubilized preparations give similar results. The use of the solubilized NaCl-pellet or the P2-pellet can considerably improve the quantitative receptor assays of plasma samples. Unlike the membrane-bound receptor, a high volume of plasma, such as 400 microliters, can be added to the assay without any influence on the 3H-DEX binding when solubilized preparation is used.

Methodological aspects of quantitative receptor assays.

Receptor assays occupy a particular position in the methods used in bioanalysis, as they do not exploit the physico-chemical properties of the analyte. These assays make use of the property of the analyte to bind to the specific binding site (receptor) and to competitively replace a labelled ligand from the same binding site. The amount of labelled ligand replaced is a measure of the amount as well as the affinity of the analyte. Thus, receptor assays offer additional information about the biological (pharmacological) activity of the analyte by distinguishing the compounds on the basis of their specific binding rather than specific molecular structure (chromatographic and non-chromatographic methods). This paper, starting with the general principles of receptor-ligand interaction, focuses on the application of ligand-binding techniques to the quantitative analysis. The factors which influence the sensitivity and the specificity of quantitative receptor assays, as well as the main directions in the improvement of the receptor preparation by using the solubilized and purified receptor are discussed. In order to enhance the use of these assays in routine practice, the development of solid-phase receptor assays is considered.

Evaluation of 3H-CGP 12177 and 3H-DHA binding to beta 2-adrenoceptors of rat reticulocytes by means of affinity spectra.

3H-CGP 12177 and 3H-DHA binding to rat reticulocyte beta 2-adrenoceptors by using an excess of 1 x 10(-5) mol l-1 propranolol was evaluated by means of affinity spectra. To describe the 3H-CGP 12177 interaction with rat reticulocytes a binding isotherm valid for one single class of specific binding sites was found to be satisfactory. On the other hand, a more complex binding isotherm had to be used for the description of the 3H-DHA interaction with rat reticulocytes. This observation further supports the preferred application of 3H-CGP 12177 over 3H-DHA for radio-receptor assay of beta-adrenoceptor blockers.