Carla A. H. Prata

Lipophilic Peptides for Gene Delivery

DNA transfections are widely performed in research laboratories and in vivo gene delivery holds the promise for curing many diseases. The synthetic carriers or vectors for DNA are typically cationic lipids. However, in biology, the recognition of nucleic acids by proteins involves both electrostatic and stacking contributions. As such we have prepared a series of new lipophilic peptide vectors that possess lysine and tryptophan amino acids for evaluation. These lipophilic peptides show minimal cytotoxicity and enhanced in vitro gene transfection activity.

Cationic Nucleoside Lipids Based on a 3-Nitropyrrole Universal Base for siRNA Delivery

Cationic nucleoside lipids based on a 3-nitropyrrole universal base were prepared from D-ribose using a straightforward chemical synthesis. Several studies including DLS, TEM, and ethidium bromide (EthBr) assay demonstrated that these amphiphilic molecules form supramolecular organizations of nanometer size in aqueous solutions and are able to bind nucleic acids. siRNA knockdown experiments were performed with these nucleolipids, and we observed protein knockdown activity similar to the siPORT NeoFX positive control. No significant cytotoxicity was found.

Cationic Nucleoside Lipids Derived from Universal Bases: A Rational Approach for siRNA Transfection

Cationic nucleoside lipids (CNLs) derived from 5-nitroindole and 4-nitroimidazole bases were prepared from d-ribose by using a straightforward chemical synthesis. TEM experiments indicate that these amphiphilic molecules self-assemble to form supramolecular organizations in aqueous solutions. Electrophoresis and standard ethidium bromide (EB) fluorescence displacement assay shows that CNLs are able to bind siRNA. We demonstrated that both the nature of the universal bases and the stereochemistry of the anomeric position (alpha, beta) have an impact on the CNLs-siRNA complex formation. Correlations among chemical structure, stereochemistry, siRNA knockdown effect, and binding affinities for all the compounds were shown and analyzed with a simple molecular modeling study. The best binding affinities for siRNA were found for the beta anomer of the 5-nitroindole CNL which exhibits protein knockdown activity similar to the standard siPORT NeoFX positive control. It is noteworthy that no significant cytotoxicity at the tested concentration was observed for the novel CNLs.

A new helper phospholipid for gene delivery.

Enhanced gene transfection activity is observed when using a new helper lipid with DOTAP, compared to DOPE.

Synthesis, characterization, and in vitro transfection activity of charge-reversal amphiphiles for DNA delivery.

A series of charge-reversal lipids were synthesized that possess varying chain lengths and end functionalities. These lipids were designed to bind and then release DNA based on a change in electrostatic interaction with DNA. Specifically, a cleavable ester linkage is located at the ends of the hydrocarbon chains. The DNA release from the amphiphile was tuned by altering the length and position of the ester linkage in the hydrophobic chains of the lipids through the preparation of five new amphiphiles. The amphiphiles and corresponding lipoplexes were characterized by DSC, TEM, and X-ray, as well as evaluated for DNA binding and DNA transfection. For one specific charge-reversal lipid, stable lipoplexes of approximately 550 nm were formed, and with this amphiphile, effective in vitro DNA transfection activities was observed.

Dendritic supramolecular assemblies for drug delivery

Dendritic supramolecular assemblies were formed in water with Reichardts dye or the anticancer drug 10-hydroxycamptothecin and the dendritic macromolecule, ([G4]-PGLSA-OH)2-PEG3400.

Supramolecular assemblies of DNA with neutral nucleoside amphiphiles

A neutral uridine-based amphiphile is described which condenses plasmid DNA. AFM studies show that the three distinct structural components of the amphiphile (i.e, nucleobase, alkyl chains, and poly(ethylene glycol)) are required for the formation of DNA-amphiphile supramolecular assemblies on a mica surface.

The effect of charge-reversal amphiphile spacer composition on DNA and siRNA delivery

A series of charge-reversal amphiphiles with different spacers separating the headgroup from the hydrophobic chains are described for delivery of DNA and siRNA. Among them, the amphiphiles possessing a glycine spacer (e.g., B-GlyGly) showed effective DNA transfection in CHO and NIH 3T3 cells, as well as siRNA gene knockdown in HepG2 and UASMC cells. Ethidium bromide quenching assays revealed that DNA was released the fastest from the lipoplex of B-GlyGly in the presence of esterase. Also, X-ray diffraction results indicated that the DNA was located between the adjacent lipid bilayers in the lipoplex of B-GlyGly. These distinct features appear to be required for high transfection activity.

Non-viral Charge Reversal Vectors for pDNA Delivery

A synthetic vector that transform from a cationic to an anionic lipid intracellularly is described. This charge-reversal lipid was synthesized and characterized, including the supramolecular complex it forms with DNA. Enhanced gene transfection was observed using this synthetic vector compared to current cationic lipids

Synthesis of Cationic Amphiphilic Surface-Block Polyester Dendrimers

AbstractCationic amphiphilic polyester dendrimers possessing varied numbers of choline and alkyl chains on the periphery were synthesized and characterized. A combination of divergent and convergent synthetic routes was used to efficiently prepare the dendrimers. All of the amphiphiles bound DNA as determined by an ethidium bromide displacement assay, and the dendrimer that contained two choline and four alkyl chains exhibited the smallest charge ratio. Only this amphiphilic dendrimer formed a well-defined structure alone or with DNA in solution, while the other dendrimer compositions gave aggregates. Specifically, small vesicular structures of several 100 nanometers in diameter were observed with DNA, and this dendrimerplex exhibited the greatest transfection efficiency of the group. The results of this study highlight the important role that charge, hydrophobicity, size, and compaction ability play in binding and formation of DNA-dendrimer complexes and the resulting transfection efficiency.Graphical AbstractCationic amphiphilic polyester dendrimers possessing different numbers of choline heads groups and myristic acid alkyl chains were synthesized and evaluated for gene transfection.