Maria C. Lima

Gemini surfactant dimethylene-1,2-bis(tetradecyldimethylammonium bromide)-based gene vectors: A biophysical approach to transfection efficiency

Cationic liposomes have been proposed as biocompatible gene delivery vectors, able to overcome the barriers imposed by cell membranes. Besides lipids, other surfactant molecules have been successfully used in the composition of gene carriers. In the present work, we used a Gemini surfactant, represented by the general structure [C(14)H(29)(CH(3))(2)N(+)(CH(2))(2)N(+)(CH(3))(2)C(14)H(29)]2Br(-) and herein designated 14-2-14, to prepare cationic gene carriers, both as the sole component and in combination with neutral helper lipids, cholesterol and DOPE. The effectiveness of three Gemini-based formulations, namely neat 14-2-14, 14-2-14:Chol (1:1 molar ratio) and 14-2-14:Chol:DOPE (2:1:1 molar ratio), to mediate gene delivery was evaluated in DNA mixtures of +/- charge ratios ranging from 1/1 to 12/1. After ruling out cytotoxicity as responsible for the differences observed in the transfection competence, structural and physical properties of the vector were investigated, using several techniques. The size and surface charge density (zeta potential) of surfactant-based structures were determined by conventional techniques and the thermotropic behaviour of aqueous dispersions of surfactant/lipid/DNA formulations was monitored by fluorescence polarization of DPH and DPH-PA probes. The capacity of lipoplexes to interact with membrane-mimicking lipid bilayers was evaluated, using the PicoGreen assay and a FRET technique. Our data indicate inefficiency of the neat 14-2-14 formulation for gene delivery, which could result from the large dimensions of the particles and/or from its relative incompetence to release DNA upon interaction with anionic lipids. The addition of cholesterol or cholesterol and DOPE conferred to Gemini-based gene carrier transfection activity at specific ranges of +/- charge ratios. Fluorescence polarization data suggest that an order parameter within a specific range was apparently needed for complexes to display maximal transfection efficiency. The transfection-competent formulations showed to be efficiently destabilized by interaction with different anionic and zwitterionic bilayers, including those containing PS and cardiolipin. These data are discussed in terms of the potential of these formulations to address different intracellular targets.

DNA pre-condensation with an amino acid-based cationic amphiphile. A viable approach for liposome-based gene delivery

A study related to the development and characterization of a new gene delivery system was performed. The approach consists in both the pre-condensation of plasmid DNA with an arginine-based cationic surfactant, arginine–N-lauroyl amide dihydrochloride (ALA), which was found not to be toxic, and the incorporation of the blood protein transferrin (Tf) into the formulations.Two cationic liposome formulations were used, one composed of a mixture of dioleoyl trimethylammoniopropane and cholesterol (DOTAP:Chol) and the other a pH sensitive formulation constituted of DOTAP, Chol, Dioleoyl phosphatidylethanolamine (DOPE) and cholesteryl hemisuccinate (CHEMS).Particles with different ALA/DNA and cationic lipid/DNA charge ratios were produced and a physicochemical characterization of the systems developed was performed. DNA conformational changes in the presence of ALA were studied by Circular Dichroism (CD) and the ALA binding to DNA was followed by surface tension measurements. Insight into the structure and morphology of the various ALA-complexes (complexes composed of ALA, DNA, Tf and liposomes) was obtained by cryogenic-Transmission Electron Microscopy (cryo-TEM) and the sizes of the ALA-complexes were determined through Photon Correlation Spectroscopy (PCS). We found that the transfection capacity of these systems is directly related with the presence of ALA and the lipidic composition. Complexes based on the pH sensitive liposome formulation present better transfection profiles. The correlation between the inner structure, density and size of the ALA-complexes and their biological activity is discussed. Overall, we demonstrate that the presence of ALA improves the transfection efficiency when conjugated with cationic liposome systems.

S4(13)-PV cell-penetrating peptide induces physical and morphological changes in membrane-mimetic lipid systems and cell membranes: implications for cell internalization.

The present work aims to gain insights into the role of peptide-lipid interactions in the mechanisms of cellular internalization and endosomal escape of the S4(13)-PV cell-penetrating peptide, which has been successfully used in our laboratory as a nucleic acid delivery system. A S4(13)-PV analogue, S4(13)-PVscr, displaying a scrambled amino acid sequence, deficient cell internalization and drug delivery inability, was used in this study for comparative purposes. Differential scanning calorimetry, fluorescence polarization and X-ray diffraction at small and wide angles techniques showed that both peptides interacted with anionic membranes composed of phosphatidylglycerol or a mixture of this lipid with phosphatidylethanolamine, increasing the lipid order, shifting the phase transition to higher temperatures and raising the correlation length between the bilayers. However, S4(13)-PVscr, in contrast to the wild-type peptide, did not promote lipid domain segregation and induced the formation of an inverted hexagonal lipid phase instead of a cubic phase in the lipid systems assayed. Electron microscopy showed that, as opposed to S4(13)-PVscr, the wild-type peptide induced the formation of a non-lamellar organization in membranes of HeLa cells. We concluded that lateral phase separation and destabilization of membrane lamellar structure without compromising membrane integrity are on the basis of the lipid-driven and receptor-independent mechanism of cell entry of S4(13)-PV peptide. Overall, our results can contribute to a better understanding of the role of peptide-lipid interactions in the mechanisms of cell-penetrating peptide membrane translocation, helping in the future design of more efficient cell-penetrating peptide-based drug delivery systems.

Partition coefficients of dopamine antagonists in brain membranes and liposomes

Partition coefficients, Kp of dopamine antagonists, spiperone, haloperidol, domperidone and pimozide were determined in caudate nucleus microsomal membranes and in liposomes from membrane lipids. Kp values were measured as a function of temperature and the thermodynamics parameters for the transfer of the drugs from the aqueous medium to the lipid bilayer were evaluated. Partition in native membranes or in liposomes formed from the membrane lipids is not strongly dependent on temperature over the range from 8 to 37 degrees. The Kp values for spiperone, haloperidol and domperidone in membrane are 32 +/- 6, 192 +/- 11 and 308 +/- 40 respectively, whereas the equivalent values in liposomes are much higher: 195 +/- 12, 558 +/- 16 and 316 +/- 16. In contrast, for pimozide, the Kp values in membranes are higher than in liposomes: 1097 +/- 11 for microsomes and 662 +/- 10 for liposomes. Partition values in natural membranes decrease sequentially as follows: pimozide greater than domperidone greater than haloperidol greater than spiperone. Membranes rich in cholesterol show lower partition coefficients for haloperidol. The interaction of the antagonists with the bilayer is associated with small enthalpy changes and large increases in entropy, as expected for hydrophobic interactions. We conclude that the partition coefficients of the drugs studied for membranes and membranes lipids are very different from those reported for octanol/water and the latter values should not be used to estimate drug partition into membranes.

Comparison of the efficiency of complexes based on S4(13)-PV cell-penetrating peptides in plasmid DNA and siRNA delivery.

The successful application of gene therapy approaches is highly dependent on the efficient delivery of nucleic acids into target cells. In the present study, new peptide-based nonviral systems were developed to enhance plasmid DNA and siRNA delivery, aiming at generating appropriate gene delivery and gene silencing tools for preclinical and clinical application. For this purpose, a new cell-penetrating peptide derived from the wild-type S4(13)-PV peptide was synthesized through the addition of a five-histidine tail to its N-terminus (H5-S4(13)-PV), and its ability to mediate gene expression and gene silencing was evaluated and compared to that of the wild-type peptide. The histidine-enriched peptide, H5-S4(13)-PV, proved to be generally more efficient and less toxic than the wild-type peptide in the delivery of plasmid DNA. In addition, complexes of H5-S4(13)-PV with siRNAs, but not of S4(13)-PV, were efficiently internalized by cells and presented high knockdown activity (63%). Interestingly, systems containing the S4(13)-PV or the H5-S4(13)-PV peptide exhibited superior biological activity when compared to those containing the reverse NLS or scrambled peptides, suggesting that both the cell-penetrating sequence and the NLS of the S4(13)-PV peptide influence the competence of binary and ternary complexes to accomplish nucleic acid delivery. In order to unravel the cancer therapeutic potential of formulations with the histidine-enriched peptide, their efficiency to mediate silencing of the oncogenic protein survivin was evaluated. As opposed to complexes with the wild-type peptide, H5-S4(13)-PV complexes showed the ability to promote a high survivin knockdown at the level of both protein (44%) and mRNA (73%), in HT1080 cells.

Safety profile of the intravenous administration of brain-targeted stable nucleic acid lipid particles.

In a clinical setting, where multiple administrations of the therapeutic agent are usually required to improve the therapeutic outcome, it is crucial to assess the immunogenicity of the administered nanoparticles. In this data work, we investigated the safety profile of the repeated intravenous administration of brain-targeted stable nucleic acid lipid particles (RVG-9r-targeted SNALPs). To evaluate local activation of the immune system, we performed analysis of mouse tissue homogenates and sections from cerebellum. To investigate peripheral activation of the immune system, we used serum of mice that were intravenously injected with RVG-9r-targeted SNALPs. These data are related and were discussed in the accompanying research article entitled “Intravenous administration of brain-targeted stable nucleic acid lipid particles alleviates Machado–Joseph disease neurological phenotype” (Conceição et al., in press) [1].

Abstract 4444: Targeted delivery of siRNAs to tumor cells and the tumor microenvironment

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL The limited effectiveness of conventional treatment strategies has generated considerable interest on the development of new types of anticancer agents, with improved molecular target specificity. In this context, gene silencing technology constitutes such new class of anticancer agents. The identification of activated oncogenes, as fundamental genetic differences relative to normal cells has made it possible to consider such genes as targets for antitumor therapy. A tumor is an organ, not a single cell type, which encompasses multiple cell types, each one contributing to the overall tumor aggressiveness. It is now recognized that novel therapeutic strategies should envisage the simultaneous inhibition of multiple targets or pathways, at different cell levels within a tumor, that regulate processes promoting tumor development. The main goal of this work was to design a novel ligand-mediated targeted lipid-based nanocarrier, containing a nucleic acid (siRNA), aiming at targeting, simultaneously, human breast cancer and endothelial cells from angiogenic vessels. We have been able to develop a novel ligand-targeted sterically stabilized lipid-based nanoparticle with adequate features for systemic administration: it is characterized by high siRNA encapsulation efficiency, efficient protection of siRNA, average size around 200 nm, and charge close to neutrality. Our results have shown that the covalent attachment of a specific ligand at the extremity of poly(ethylene glycol) chains, brings a major advantage as it significantly improves the internalization of the lipid-based nanoparticle by both human cancer cells (MDA-MB-435 and MDA-MB-231) and endothelial cells from angiogenic blood vessels (HMEC-1). Moreover, it was not observed a significant internalization by the non-transformed cell line, BJ fibroblasts, indicating the cellular specificity of the developed targeted nanoparticle. In order to evaluate the potential of the developed targeted nanoparticle to silence a target gene, the green fluorescent protein (GFP)-overexpressing MDA-MB-435 cells were used. A specific downregulation of GFP, both at the protein and mRNA levels, was further observed with the targeted nanoparticle when compared with the non-targeted counterpart. As the developed nanoparticle is adequate for the encapsulation and delivery of any siRNA sequence, studies with a siRNA targeting a validated molecular target are now ongoing. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4444. doi:10.1158/1538-7445.AM2011-4444