Isabel S. Oliveira

Abstract Polymeric hydrogels are traditionally employed as drug reservoirs in topical delivery, but they can also function as scaffolds for drug-loaded nanocarriers, enabling hybrid systems with enhanced performance. In this work, we report a thermo-adaptive hybrid hydrogel-composed of a block copolymer scaffold and a network of surfactant-based nano- and microtubes-which exhibits a mechanism herein termed inversely coupled thermogelation (ICT). The scaffold consists of Pluronic F127, a biocompatible triblock copolymer that transitions from micellar solution to a cubic liquid crystalline gel upon heating. The tubular network arises from the self-assembly of biomimetic lysine-derived surfactants. Crucially, when the block copolymer/surfactant hybrid is heated from 20 degrees C to 35 degrees C (approx. skin temperature), the surfactant tubes disassemble into micelles or vesicles, while the block copolymer forms the cubic phase. Accordingly, a tube-dominated gel evolves into a block copolymer-dominated gel through a gel-solution-gel sequence uniquely driven by the opposing thermal responses of the two constituents. This results in a hybrid system that is not only spreadable, self-healing, and mechanically robust, but also well-suited for sustained topical delivery. Imaging, calorimetry, and rheology provide detailed insights into the structure, phase transitions, and flow behavior of the hybrid system and its individual components. As a proof-of-concept, the gel enables slow, sustained release of a fluorescent model probe (carboxyfluorescein), exhibits excellent cytocompatibility, and promotes high cell internalization. Overall, this ICT-based strategy establishes a versatile and sustainable platform with strong potential for long-term topical drug delivery.

Abstract Catanionic mixtures, composed of cationic and anionic surfactants, spontaneously form robust self-assembled aggregates whose morphology, size, and surface charge can be tailored by adjusting the surfactant mixing ratio. This straightforward and scalable approach, based on easily obtainable components, offers a versatile and simple platform with high potential for drug delivery. However, developing viable nanocarriers also requires a favorable cytotoxicity profile, high drug loading, and strong bioactivity-features that catanionic vesicles often lack. Here, we present a systematic study of pH-sensitive catanionic vesicles composed of mixtures of the biocompatible, FDA-approved anionic surfactant sodium lauroyl sarcosinate (SLSar) and various cationic double-tailed surfactants (didodecyldimethylammonium bromide and bis-quat 12-s-12 gemini surfactants). The different vesicle systems form spontaneously at low critical aggregation concentrations (approximate to 3-30 mu mol kg-1), and exhibit a broad range of size distributions, high surface charge (positive and negative), and long-term colloidal stability. Cytotoxicity screening in healthy L929 fibroblasts enabled the selection of highly biocompatible compositions, with gemini/SLSar systems showing superior doxorubicin (DOX) encapsulation efficiency. These vesicles exhibit enhanced DOX release at acidic pH (approximate to 6.0), mimicking tumor microenvironments, and demonstrate rapid and efficient uptake in lung carcinoma cells within 30 min, increasing over 3 h. Remarkably, DOX-loaded vesicles achieve potent cytotoxicity at only 5 nM DOX-well below the IC50 of free drug-highlighting enhanced therapeutic efficacy and potential for reduced systemic toxicity. Overall, SLSar-based catanionic vesicles constitute a simple, stable, and tunable nanocarrier platform with significant potential for pH-responsive, low-dose cancer chemotherapy.

Abstract The efficient delivery of nucleic acids (NAs) remains a major challenge in gene therapy due to their poor stability and limited cellular uptake. Even though non-viral vectors have been pivotal to overcoming some of these challenges, significant barriers, such as intracellular digestion of NAs and limited endosomal escape, still remain. Here, we developed novel stimuli-responsive lipoplexes integrating a 2-hydroxychalcone-based cationic amphiphile (CnNCh, with 4 or 6 carbons in their alkyl chains, n = 4 or 6) and monoolein (MO). This combination leverages the photoisomerization and pH-sensitivity of chalcone derivatives, along with the fusogenic capabilities of MO, to achieve enhanced transfection efficiency via light irradiation. To reach this goal, we first assessed the cytotoxicity of the cationic amphiphiles in healthy and tumor cells. We then prepared mixtures with varying CnNCh/MO molar ratios, yielding net cationic vesicles with long-term colloidal stability. Subsequently, NAs were efficiently compacted into lipoplexes at N/P ratios (positively charged nitrogen/negatively charged phosphate) higher than 1, attaining near-complete compaction. Light and pH stimuli induce the formation of the expected products, but without compromising lipoplex stability or activating premature NA release. Vesicles with different CnNCh/MO molar ratios do not induce the loss of viability of normal fibroblasts for concentrations up to 50 mu M. Crucially, siRNA-lipoplex mixtures having C4NCh/MO molar ratios of 1/1 and 2/1 (N/P = 6) achieve significant GFP knockdown after irradiation, indicative of successful siRNA delivery and biological effects. Using biomimicking endosomal membranes, we show that photoactivation enhances membrane fusion, suggesting a mechanism entailing light-mediated endosomal escape. Our study provides proof-of-concept for a light-switch mechanism offering precise spatiotemporal control over gene silencing, a highly desirable feature in therapeutic applications.

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.