Robert Fuks

The role of endosome destabilizing activity in the gene transfer process mediated by cationic lipids

We used a 32P‐labeled pCMV‐CAT plasmid DNA to estimate the DNA uptake efficiency and unlabeled pCMV‐CAT plasmid DNA to quantify the CAT activity after transfection of COS cells using each of the three following cationic compounds: [1] vectamidine (3‐tetradecylamino‐N‐tert‐butyl‐N′‐tetradeccyl‐propionamidine, and previously described as diC14‐amidine [1]), [2] lipofectin (a 1:1 mixture of N‐(1‐2,3‐dioleyloxypropyl)N,N,N‐triethylammonium (DOTMA) and dioleylphosphatid‐ylethanolamine (DOPE)), and [3] DMRIE‐C (a 1:1 mixture of N‐[1‐(2,3‐dimyristyloxy)propyl]‐N,N‐dimethyl‐N‐(2‐hydroxyethyl) ammonium bromide (DMRIE) and cholesterol). Surprisingly, a high CAT activity was observed with vectamidine although the DNA uptake efficiency was lower as compared to lipofectin and DMRIE‐C. Transmission electron microscopy (TEM) revealed endocytosis as the major pathway of DNA‐cationic lipid complex entry into COS cells for the three cationic lipids. However, the endosomal membrane in contact with complexes containing vectamidine or DMRIE‐C often exhibited a disrupted morphology. This disruption of endosomes was much less frequently observed with the DNA‐lipofectin complexes. This comparison of the three compounds demonstrate that efficient transfection mediated by cationic lipids is not only correlated to their percentage of uptake but also to their ability to destabilize and escape from endosomes.

INTRACELLULAR VISUALIZATION OF BRDU-LABELED PLASMID DNA/CATIONIC LIPOSOME COMPLEXES

Difficulties in specific detection of transfected DNA in cells represent an important limitation in the study of the gene transfer process. We studied the cellular entry and fate of a plasmid DNA complexed with a cationic lipid, Vectamidine (3-tetradecyl-amino-N-tert-butyl-N-tetradecylpropionamidine) in BHK21 cells. To facilitate its detection inside the cells, bromodeoxyuridine (BrdU) was incorporated into plasmid DNA under conditions that minimize plasmid alteration. BrdU was localized in cells incubated with Vectamidine/BrdU-labeled plasmid DNA complexes by immunogold labeling and electron microscopy (EM). Labeling was predominantly associated with aggregated liposome structures at the surface of and inside the cells. EM observations of cells transfected with Vectamidine/DNA complexes showed that the liposome/DNA aggregates accumulate in large vesicles in the cell cytosol. On the other hand, using rhodamine-labeled Vectamidine and revealing BrdU with FITC-conjugated antibodies permitted simultaneous detection in the cells of both components of the complexes with confocal laser scanning microscopy. The DNA and lipids co-localized at the surface of and inside the cells, indicating that the complex is internalized as a whole. Our results show that the BrdU-labeled plasmid DNA detection system can be a useful tool to visualize exogenous DNA entry into cells by a combination of electron and confocal microscopy.

Double long-chain amidine liposome-mediated self replicating DNA transfection

We present experimental evidence that a complex made of a double long chain cationic amphiphile and recombinant mRNA facilitates the entry and expression of genetic material into cells. Combining the properties of the self replicating recombinant mRNA driven by the Semliki Forest Virus (SFV) replicon and the transfection potentialities of a new cationic amphiphile (N‐t‐butyl‐N′‐tetradecyl‐3‐tetradecylaminopropionamidine) yields a highly efficient mRNA transfection system conferring up to 100% infectivity. The preparation and characterization of the long chain amidine cationic amphiphile‐mRNA complex as well as the influence of the diC14‐amidine/RNA ratio on the infective activity are described.

Specific adsorption of serine proteases on coated silica beads substituted with amidine derivatives

Amidine derivatives interact with serine proteases, the inhibition being due to interactions between amidine functions and the active sites of the enzymes. Five different types of amidine (substituted or unsubstituted) were coupled to coated silica beads, which had previously been coated with DEAE-dextran to minimize the non-specific interactions due to silanol groups. Coated silica functionalized with substituted amidines shows a strong affinity towards human plasmin. This affinity is probably due to hydrophobic interactions between the substituted amidine and the human plasmin structure. Coated silica grafted by p-aminobenzamide gives a specific interaction with human plasmin. The importance of ionic strength and the steric conformation of the ligand is discussed. This support was used to purify thrombin from crude preparations by high-performance affinity chromatography.

Vesicle formation by double long-chain amidines

The formation and characterization of synthetic surfactant vesicles prepared from aqueous suspensions of a new class of amphiphiles with an amidine function as the polar head group are reported.

Synthesis of N-Substituted α-Halo-Propenamidines

Abstract The synthesis of the not yet described title compounds has been carried out to study the influence of the halogen atom on various properties of propenamidines. The α-chloro derivatives 3 have been prepared from the commercially available α-chlo-ro-propenenitrile 1 by amination of its nitrilium tetrachloroferrate 2.

Détermination de constantes d'acidité d'amidines N-N′-substituées. Relations entre les pKA et les vibrations IR — I. Acétamidines et métacrylamidines

Abstract Changes in the ultraviolet absorption spectra with pH in aqueous solution were used to determine the pK A values of amidines. A new mathematical procedure was used to study sparingly soluble compounds. In this technique, all the experimental data are used to solve for the absorbance of the insoluble basic species and for the pK A value by a ‘complex method’ of optimization, which is more general than the classical least-squares method. A relationship between a double bond vibration frequency and the acidity constants of these compounds has been demonstrated.

The Michael Addition of Enamines to Propenamidines. A Convenient Synthesis of 2β-Amidinoethylcyclo-Alkanones by Amidinoethylation Reaction

Abstract A variety of propenamidines 3 have been reacted with enamines of cyclopentanone and cyclohexanone giving after hydrolysis of the Stork enamine 6, 2-β-amidinoethylcyclopentanones 8 and -hexanones 7. This reaction illustrates the electrophilic character of the C=C double bond due to the conjugated amidine function, thus providing with propenamidines 3 a new class of Michael acceptors for enamines.