María del Carmen Morán

Monoglyceride surfactants from arginine: synthesis and biological properties

A novel family of dicationic arginine-monoglyceride surfactants, 1-acyl-3-O-(L-arginyl)-rac-glycerol·2HCl, was synthesised and characterised. They have one alkyl chain of with length in the range of C10–C14 attached to the glycerol though esters bonds and a dicationic polar head from the arginine. Structurally they can be regarded as analogues of the monoglycerides, widely used as emulsifiers in the food and in the pharmaceutical industry. The introduction of the basic amino acid arginine into the monoglycerides increases the solubility of these compounds and improves their antimicrobial activity. Moreover, the acute toxicity of these surfactants against Daphnia magna is clearly lower than the toxicity reported for conventional cationic surfactants.

Gelatin-based nanoparticles as DNA delivery systems: Synthesis, physicochemical and biocompatible characterization

The rapidly rising demand for therapeutic grade DNA molecules requires associated improvements in encapsulation and delivery technologies. One of the challenges for the efficient intracellular delivery of therapeutic biomolecules after their cell internalization by endocytosis is to manipulate the non-productive trafficking from endosomes to lysosomes, where degradation may occur. The combination of the endosomal acidity with the endosomolytic capability of the nanocarrier can increase the intracellular delivery of many drugs, genes and proteins, which, therefore, might enhance their therapeutic efficacy. Among the suitable compounds, the gelification properties of gelatin as well as the strong dependence of gelatin ionization with pH makes this compound an interesting candidate to be used to the effective intracellular delivery of active biomacromolecules. In the present work, gelatin (either high or low gel strength) and protamine sulfate has been selected to form particles by interaction of oppositely charged compounds. Particles in the absence of DNA (binary system) and in the presence of DNA (ternary system) have been prepared. The physicochemical characterization (particle size, polydispersity index and degree of DNA entrapment) have been evaluated. Cytotoxicity experiments have shown that the isolated systems and the resulting gelatin-based nanoparticles are essentially non-toxic. The pH-dependent hemolysis assay and the response of the nanoparticles co-incubated in buffers at defined pHs that mimic extracellular, early endosomal and late endo-lysosomal environments demonstrated that the nanoparticles tend to destabilize and DNA can be successfully released. It was found that, in addition to the imposed compositions, the gel strength of gelatin is a controlling parameter of the final properties of these nanoparticles. The results indicate that these gelatin-based nanoparticles have excellent properties as highly potent and non-toxic intracellular delivery systems, rendering them promising DNA vehicles to be used as non-viral gene delivery systems.

Lysine-based surfactants in nanovesicle formulations: the role of cationic charge position and hydrophobicity in in vitro cytotoxicity and intracellular delivery

Abstract Understanding nanomaterial interactions within cells is of increasing importance for assessing their toxicity and cellular transport. Here, the authors developed nanovesicles containing bioactive cationic lysine-based amphiphiles and assessed whether these cationic compounds increase the likelihood of intracellular delivery and modulate toxicity. Different cytotoxic responses were found among the formulations, depending on surfactant, cell line and endpoint assayed. The induction of mitochondrial dysfunction, oxidative stress and apoptosis were the general mechanisms underlying cytotoxicity. Fluorescence microscopy analysis demonstrated that nanovesicles were internalised by HeLa cells and evidenced that their ability to release endocytosed materials into cell cytoplasm depends on the structural parameters of amphiphiles. The cationic charge position and hydrophobicity of surfactants determine the nanovesicle interactions within the cell and, thus, the resulting toxicity and intracellular behaviour after cell uptake of the nanomaterial. The insights into some toxicity mechanisms of these new nanomaterials contribute in reducing the uncertainty surrounding their potential health hazards.

Mixed protein-DNA gel particles for DNA delivery: Role of protein composition and preparation method on biocompatibility

Mixtures of two cationic proteins were used to prepare protein-DNA gel particles, employing associative phase separation and interfacial diffusion (Morán et al., 2009a). By mixing the two proteins, we have obtained particles that displayed higher loading efficiency and loading capacity values than those obtained in single-protein systems. However, nothing is known about the adverse effects on haemocompatibility and cytotoxicity of these protein-DNA gel particles. Here, we examined the interaction of protein-DNA gel particles obtained by two different preparation methods, and their components, with red blood cells and established cells. From a haemolytic point of view, these protein-DNA gel particles were demonstrated to be promising long-term blood-contacting medical devices. Safety evaluation with the established cell lines revealed that, in comparison with proteins in solution, the cytotoxicity was reduced when administered in the protein-DNA systems. In comparison with large-sized particles, the cytotoxic responses of small-sized protein-DNA gel particles showed to be strongly dependent of both the protein composition and the cell line being the tumour cell line HeLa more sensitive to the deleterious effects of the mixed protein-based particles. The observed trends in haemolysis and cell viabilities were in agreement with the degree of complexation values obtained for the protein-DNA gel particles prepared by both preparation methods.

Oil-in-water nanoemulsions are suitable for carrying hydrophobic compounds: Indomethacin as a model of anti-inflammatory drug

Oil-in-water nanoemulsions are increasingly being used as delivery systems for encapsulating lipophilic components in functional food, personal care and pharmaceutical products. In the current study, we developed a multimodal platform to carry hydrophobic indomethacin or magnetic nanoparticles, or both. As a consequence, this platform has great potential for therapeutic or imaging purposes. By optimizing the system composition and homogenization conditions, a nanoemulsion with a mean droplet diameter of about 200nm and a low polydispersity index (<0.2) was formed. The plain nanoemulsion was shown to be innocuous in cellular studies and did not present acute toxicity (observed in a rat model). More interesting was the finding that nanoemulsions loaded with indomethacin presented a significantly different anti-inflammatory than the free drug.

Effect of PEGylation on Ligand-Targeted Magnetoliposomes: A Missed Goal

We tested the targeting efficiency of magnetoliposomes (MLPs) labeled with tripeptide arginine-glycine-aspartic acid (RGD) on two types of cells: HeLa cells expressing RGD receptors and 3T3 cells lacking RGD receptors. The targeting ability of RGD-MLPs was compared to that of bare MLPs and MLPs stabilized with poly(ethylene glycol) (PEG). Cellular internalization of these liposomes was determined by flow cytometry and confocal microscopy, which showed that both types of cells took up more nontargeting MLPs than targeting RGD-MLPs or PEG-MLPs, with PEG-MLPs showing the lowest degree of internalization. The presence of specific receptors on HeLa cells did not facilitate the binding of RGD-MLPs, probably due to the presence of PEG chains on the liposomal surface. The polymer increases the circulation time of the liposomes in the organism but reduces their interactions with cells. Despite the localization of the RGD peptide on the tip of PEG in RGD-MLPs, the interaction between the liposome and cell was still limited. To avoid this drawback, targeting drug delivery systems can be prepared with two types of PEG: one of a short length to enable biocompatibility and the other of a longer chain to carry the ligand.