Publications

Abstract Malaria is one of the big three global infectious diseases, having caused above two hundred million cases and over half a million deaths in 2020. The continuous demand for new treatment options prioritizes the cost-effective development of new chemical entities with multi-stage antiplasmodial activity, for higher efficacy and lower propensity to elicit drug-resistant parasite strains. Following up on our long-term research towards the rescue of classical antimalarial aminoquinolines like chloroquine and primaquine, we have developed new organic salts by acid-base pairing of those drugs with natural bile acids. These antimalarial drug-derived bile salts were screened in vitro against the hepatic, blood and gametocyte stages of Plasmodium parasites, unveiling chloroquine bile salts as unprecedented triple-stage antiplasmodial hits. These findings pave a new pathway for drug rescuing, even beyond anti-malarial and other anti-infective drugs. Malaria is one of the big three global infectious diseases, with the heaviest toll on human lives in low-to-middle income countries. Cost-effective antimalarial drugs with multi-stage action remain an unmet and urgent need in global healthcare.

Abstract Cationic gemini surfactants have emerged as potential gene delivery agents as they can co-assemble with DNA due to a strong electrostatic association. Commonly, DNA complexation is enhanced by the inclusion of a helper lipid (HL), which also plays a key role in transfection efficiency. The formation of lipoplexes, used as non-viral vectors for transfection, through electrostatic and hydrophobic interactions is affected by various physicochemical parameters, such as cationic surfactant:HL molar ratio, (+/-) charge ratio, and the morphological structure of the lipoplexes. Herein, we investigated the DNA complexation ability of mixtures of serine-based gemini surfactants, (nSer)2N5, and monoolein (MO) as a helper lipid. The micelle-forming serine surfactants contain long lipophilic chains (12 to 18 C atoms) and a five CH2 spacer, both linked to the nitrogen atoms of the serine residues by amine linkages. The (nSer)2N5:MO aggregates are non-cytotoxic up to 35-90 mu M, depending on surfactant and surfactant/MO mixing ratio, and in general, higher MO content and longer surfactant chain length tend to promote higher cell viability. All systems efficaciously complex DNA, but the (18Ser)2N5:MO one clearly stands as the best-performing one. Incorporating MO into the serine surfactant system affects the morphology and size distribution of the formed mixed aggregates. In the low concentration regime, gemini-MO systems aggregate in the form of vesicles, while at high concentrations the formation of a lamellar liquid crystalline phase is observed. This suggests that lipoplexes might share a similar bilayer-based structure.

Abstract Mixtures of cationic and anionic surfactants often originate bilayer structures, such as vesicles and lamellar liquid crystals, that can be explored as model membranes for fundamental studies or as drug and gene nanocarriers. Here, we investigated the aggregation properties of two catanionic mixtures containing biomimetic surfactants derived from serine. The mixtures are designated as 12Ser/8-8Ser and 14Ser/10-10Ser, where mSer is a cationic, single-chained surfactant and n-nSer is an anionic, double-chained one (m and n being the C atoms in the alkyl chains). Our goal was to investigate the effects of total chain length and chain length asymmetry of the catanionic pair on the formation of catanionic vesicles, the vesicle properties and the vesicle/micelle transitions. Ocular observations, surface tension measurements, video-enhanced light microscopy, cryogenic scanning electron microscopy, dynamic and electrophoretic light scattering were used to monitor the self-assembly process and the aggregate properties. Catanionic vesicles were indeed found in both systems for molar fractions of cationic surfactant >= 0.40, always possessing positive zeta potentials (zeta = +35-50 mV), even for equimolar sample compositions. Furthermore, the 14Ser/10-10Ser vesicles were only found as single aggregates (i.e., without coexisting micelles) in a very narrow compositional range and as a bimodal population (average diameters of 80 and 300 nm). In contrast, the 12Ser/8-8Ser vesicles were found for a wider sample compositional range and as unimodal or bimodal populations, depending on the mixing ratio. The aggregate size, pH and zeta potential of the mixtures were further investigated. The unimodal 12Ser/8-8Ser vesicles (<D-H> approximate to 250 nm, pH approximate to 7-8, zeta approximate to +32 mV and a cationic/anionic molar ratio of approximate to 2:1) are particularly promising for application as drug/gene nanocarriers. Both chain length asymmetry and total length play a key role in the aggregation features of the two systems. Molecular insights are provided by the main findings.

Abstract Interactions between polymers (P) and surfactants (S) in aqueous solution lead to interfacial and aggregation phenomena that are not only of great interest in physical chemistry but also important for many industrial applications, such as the development of detergents and fabric softeners. Here, we synthesized two ionic derivatives-sodium carboxymethylcellulose (NaCMC) and quaternized cellulose (QC)-from cellulose recycled from textile wastes and then explored the interactions of these polymers with assorted surfactants-cationic (CTAB, gemini), anionic (SDS, SDBS) and nonionic (TX-100)-commonly used in the textile industry. We obtained surface tension curves of the P/S mixtures by fixing the polymer concentration and then increasing the surfactant concentration. In mixtures where polymer and surfactant are oppositely charged (P-/S+ and P+/S-), a strong association is observed, and from the surface tension curves, we determined the critical aggregation concentration (cac) and critical micelle concentration in the presence of polymer (cmc(p)). For mixtures of similar charge (P+/S+ and P-/S-), virtually no interactions are observed, with the notable exception of the QC/CTAB system, which is much more surface active than the neat CTAB. We further investigated the effect of oppositely charged P/S mixtures on hydrophilicity by measuring the contact angles of aqueous droplets on a hydrophobic textile substrate. Significantly, both P-/S+ and P+/S- systems greatly enhance the hydrophilicity of the substrate at much lower surfactant concentrations than the surfactant alone (in particular in the QC/SDBS and QC/SDS systems).

Abstract Inspired by previous disclosure of room-temperature ionic liquids derived from primaquine and cinnamic acids, which displayed slightly enhanced blood-stage activity compared to the parent drug, we have now combined this emblematic antimalarial with natural fatty acids. This affords surface-active ionic liquids whose liver-stage antiplasmodial activity is either retained or slightly enhanced, while revealing blood-stage antiplasmodial activity at least one order of magnitude higher than that of the parent compound. These findings open new perspectives towards the cost-effective recycling of classical drugs that are either shelved or in decline, and which is not limited to antimalarial agents.

Abstract Aqueous surfactant/polymermixtures form colloidal structures of great fundamental interest and practical relevance, such as nanostructured hydrogels for biomedical and pharmaceutical uses. In this work, we investigated the phase behavior, structure and cytotoxicity of mixtures of a double-tailed lysine-based surfactant, 16Lys12, and two hydroxyethyl cellulose (HEC) derivatives, JR400 and LM200. The surfactant, S-, is anionic and self-assembles into tubular structures at room temperature, undergoing a tubule-to-vesicle transition at approximate to 44 degrees C. JR400 is a cationic homopolymer, P+, longer and more densely charged than LM200, a closely related hydrophobically modified polymer, HMP+. Electrostatic and hydrophobic interactions play a crucial role in the observed phase behavior and resulting colloidal structures. Both the S-/P+ and S-/HMP+ mixtures show three main phase regions: at surfactant charge excess, bluish dispersions containing mixed polymer/tubular aggregates and, upon heating, polymer/vesicle clusters; a white precipitate near charge equimolarity, coexisting with either a solution or a gel; and highly viscous hydrogels, at polymer charge excess. In the bluish dispersions, the S-/P+ and S-/HMP+ systems show relevant differences in thermal behavior and type of aggregates present. Cryogenic scanning electron microscopy shows that the hydrogels consist of honeycomb-like structure of surfactant and polymer moieties. Cytotoxicity assays in the bluish dispersion region indicate good levels of cytocompatibility for both types of surfactant/polymer systems. Overall, these dispersions and hydrogels can be further explored for the encapsulation and temperature-triggered release of biomolecules.

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 In this work, we explore the ability of newly synthesized threonine-derived surfactants to form robust, versatile and cytocompatible catanionic vesicles when mixed with gemini surfactants, as potential effective nanocarriers for biomolecules. The threonine surfactants consist of single-tailed amphiphiles with carboxylate headgroups and varying alkyl tail length, CnThr, where n is the (even) number of tail C atoms, varying from 8 to 16. After an initial characterization of the micellization behavior of the neat CnThr surfactants (at pH = 7 and 12), the dodecyl derivative, C12Thr, was selected as the optimal surfactant to investigate regions of formation of spontaneous catanionic vesicles. Phase behavior studies and microstructural characterization of mixtures involving both conventional bis-quat n-s-n gemini (where n and s are the tail and spacer number of C atoms) and biocompatible serine-derived gemini surfactants were carried out. Light and electron microscopy, dynamic light scattering and zeta potential measurements show spontaneous vesicles indeed form and exhibit versatile features in terms of average size, morphology, polydispersity, surface charge and pH. The toxicological profile of the neat surfactants and C12Thr/gemini vesicles based on MTT assays with a L929 cell line was also evaluated, showing good levels of in vitro cytocompatibility. Overall, the assortment of developed catanionic vesicles offers very attractive physicochemical and biological features to be explored for delivery purposes.

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.