Isabel S. Oliveira

Abstract Non-viral gene therapy based on gene silencing with small interfering RNA (siRNA) has attracted great interest over recent years. Among various types of cationic complexation agents, amino acid-based surfactants have been recently explored for nucleic acid delivery due to their low toxicity and high biocompatibility. Monoolein (MO), in turn, has been used as helper lipid in liposomal systems due to its ability to form inverted nonbilayer structures that enhance fusogenicity, thus contributing to higher transfection efficiency. In this work, we focused on the development of nanovectors for siRNA delivery based on three gemini amino acid-based surfactants derived from serine - (12Ser)(2)N12, amine derivative; (12Ser)(2)COO12, ester derivative; and (12Ser)(2)CON12, amide derivative - individually combined with MO as helper lipid. The inclusion of MO in the cationic surfactant system influences the morphology and size of the mixed aggregates. Furthermore, the gemini surfactant:MO systems showed the ability to efficiently complex siRNA, forming stable lipoplexes, in some cases clearly depending on the MO content, without inducing significant levels of cytotoxicity. High levels of gene silencing were achieved in comparison with a commercially available standard indicating that these gemini:MO systems are promising candidates as lipofection vectors for RNA interference (RNAi)-based therapies.

Abstract Drug delivery vectors based on amphiphiles have important features such as versatile physicochemical properties and stimuli-responsiveness. Amino acid-based surfactants are especially promising amphiphiles due to their enhanced biocompatibility compared to conventional surfactants. They can self-organize into micelles, vesicles and complex hierarchical structures, such as fibers, twisted and coiled ribbons, and tubules. In this work, we investigated the self-assembly and drug loading properties of a family of novel anionic double-tailed lysine-derived surfactants, with variable degree of tail length mismatch, designated as mLys10 and 10Lysn, where m and n are the number of carbon atoms in the tails. These surfactants form tubular aggregates with assorted morphologies in water that undergo gelation due to dense entanglement, as evidenced by light and electron microscopy. Lysozyme (LZM), an enzyme with antimicrobial properties, was selected as model protein for loading. After the characterization of the interfacial properties and phase behavior of the amphiphiles, the LZM-loading ability of the tubules was investigated, under varying experimental conditions, to assess the efficiency of the aggregates as pH- and temperature-sensitive nanocarriers. Further, the toxicological profile of the surfactants per se and surfactant/LZM hydrogels was obtained, using human skin fibroblasts (BJ-5ta cell line). Overall, the results show that the tubule-based hydrogels exhibit very interesting properties for the transport and controlled release of molecules of therapeutic interest.

Abstract Ionic liquids derived from classical antimalarials are emerging as a new approach towards the cost-effective rescuing of those drugs. Herein, we disclose novel surface-active ionic liquids derived from chloroquine and natural fatty acids whose antimalarial activity in vitro was found to be superior to that of the parent drug. The most potent ionic liquid was the laurate salt of chloroquine, which presented IC(50)values of 4 and 110 nM against a chloroquine-sensitive and a chloroquine-resistant strain ofPlasmodium falciparum, respectively, corresponding to an 11- and 6-fold increase in potency as compared to the reference chloroquine bisphosphate salt against the same strains. This unprecedented report opens new perspectives in both the fields of malaria chemotherapy and of surface-active ionic liquids derived from active pharmaceutical ingredients.

Abstract Dioctadecyldimethylammonium bromide (DODAB):Monoolein (MO) lipoplexes have mainly been studied within the range of high molar ratios of DODAB, with noticeable transfection efficiencies in the Human Embryonic Kidney (HEK, a.k.a. 293T) cell line. In this work, we intend to study the effect of high MO content on the structure and physicochemical properties of pDNA/DODAB:MO lipoplexes to achieve some correlation with their transfection efficiency. Static/Dynamic Light Scattering and Cryo-TEM imaging were used to characterize the size/morphology of DNA/DODAB:MO lipoplexes at different DODAB:MO contents (2:1, 1:1, 1:2) and charge ratios (CRs) (+/-). Nile Red fluorescence emission was performed to detect changes in microviscosity, hydration and polarity of DNA/DODAB:MO systems. Lipoplexes stability at physiological pH values and in the presence of anionic lipids was evaluated by Forster Resonance Energy Transfer (FRET). Physicochemical/structural data were complemented with transfection studies in HEK cells using the beta-galactosidase reporter gene activity assay. This work reports the coexistence of multilamellar and non-lamellar inverted phases in MO-richer lipoplexes (DODAB:MO 1:2 and 1:4), leading to transfection efficiencies comparable to those of multilamellar (DODAB-richer) lipoplexes, but at higher charge ratios [CR (+/-) = 6.0] and without dose-effect response. These results may be related to the structural changes of lipoplexes promoted by high MO content.

Abstract A recently described non-viral gene delivery system [dioctadecyldimethylammonium bromide (DODAB)/monoolein (MO)] has been studied in detail to improve knowledge on the interactions between lamellar (DODAB) and non-lamellar-forming (MO) lipids, as a means to enhance their final cell transfection efficiency. Indeed, the morphology, fluidity, and size of these cationic surfactant/neutral lipid mixtures play an important role in the ability of these systems to complex nucleic acids. The different techniques used in this work, namely dynamic light scattering (DLS), fluorescence spectroscopy, differential scanning calorimetry (DSC), cryogenic transmission electron microscopy (cryo-TEM), light microscopy (LM), and surface pressure-area isotherms, allowed fully characterization of the phase behavior and aggregate morphology of DODAB/MO mixtures at different molar ratios. Overall, the results indicate that the final morphology of DODAB/MO aggregates depends on the balance between the tendency of DODAB to form zero-curvature bilayer structures and the propensity of MO to form non-bilayer structures with negative curvature. These results also show that in the MO-rich region, an increase in temperature has a similar effect on aggregate morphology as an increase in MO concentration.