Formation of catanionic vesicles by threonine-derived surfactants and gemini surfactants based on conventional or serine-derived headgroups: designing versatile and cytocompatible nanocarriers.

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.

Size, charge, and stability of fully serine-based catanionic vesicles: towards versatile biocompatible nanocarriers.

Vesicles based on mixed cationic and anionic surfactants (catanionic vesicles) offer a number of advantageous colloidal features over conventional lipid-based vesicles, namely spontaneity in formation, long-term stability, and easy modulation of size and charge. If biocompatibility is added through rational design of the chemical components, the potential for biorelated applications further emerges. Here, we report for the first time on two catanionic vesicle systems in which both ionic amphiphiles are derivatized from the same amino acid--serine--with the goal of enhancing aggregate biocompatibility. Phase behavior maps for a mixture with chain length symmetry, 12Ser/12-12Ser, and another with asymmetry, 16Ser/8-8Ser, are presented, for which regions of vesicles, micelles, and coexisting aggregates are identified. For the asymmetric mixture, detailed phase behavior and microstructure characterization have been carried out based on surface tension, light microscopy, cryo-SEM, cryo-TEM, and dynamic light scattering analysis. Vesicles are found with tunable mean size, pH, and zeta potential. Changes in aggregate shape with varying composition and the effect of preparation methods and aging on vesicle features and stability have been investigated in detail. The results are discussed in the light of self-assembly models and related catanionic systems reported before. A versatile system of robust vesicles is thus presented for potential applications.

Gemini surfactants mediate efficient mitochondrial gene delivery and expression.

Gene delivery targeting mitochondria has the potential to transform the therapeutic landscape of mitochondrial genetic diseases. Taking advantage of the nonuniversal genetic code used by mitochondria, a plasmid DNA construct able to be specifically expressed in these organelles was designed by including a codon, which codes for an amino acid only if read by the mitochondrial ribosomes. In the present work, gemini surfactants were shown to successfully deliver plasmid DNA to mitochondria. Gemini surfactant-based DNA complexes were taken up by cells through a variety of routes, including endocytic pathways, and showed propensity for inducing membrane destabilization under acidic conditions, thus facilitating cytoplasmic release of DNA. Furthermore, the complexes interacted extensively with lipid membrane models mimicking the composition of the mitochondrial membrane, which predicts a favored interaction of the complexes with mitochondria in the intracellular environment. This work unravels new possibilities for gene therapy toward mitochondrial diseases.

Serine-based gemini surfactants with different spacer linkages: from self-assembly to DNA compaction.

Cationic gemini surfactants have strong potential as compaction agents of nucleic acids for efficient non-viral gene delivery. In this work, we present the aggregation behavior of three novel cationic serine-based gemini surfactants as well as their ability to compact DNA per se and mixed with a helper lipid, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). All the surfactants have a 12-12-12 configuration, i.e. two main 12-carbon alkyl chains linked to the nitrogen atom of the amino acid residue and a 12 methylene spacer, but they differ in the nature of the spacer linkage: for (12Ser)2N12, an amine bond; for (12Ser)2CON12, an amide bond; and for (12Ser)2COO12, an ester bond. Interestingly, while the amine-based gemini aggregates into micelles, the amide and ester ones spontaneously form vesicles, which denotes a strong influence of the type of linkage on the surfactant packing parameter. The size, ζ-potential and stability of the vesicles have been characterized by light microscopy, cryogenic scanning electron microscopy (cryo-SEM) and dynamic light scattering (DLS). The interaction of the gemini aggregates with DNA at different charge ratios and in the absence and presence of DOPE has been studied by DLS, fluorescence spectroscopy and cryo-SEM. All the compounds are found to efficiently compact DNA (complexation > 90%), but relevant differences are obtained in terms of the size, ζ-potential and stability of the lipoplexes formed. Results are rationalized in terms of headgroup differences and the type of aggregates present prior to DNA condensation.

Serine-based surfactants as effective antimicrobial agents against multiresistant bacteria.

The antimicrobial activity of two serine derived gemini cationic surfactants, amide (12Ser)2CON12 and ester (12Ser)2COO12, was tested using sensitive, E. coli ATCC 25922 and S. aureus ATCC 6538, and resistant, E. coli CTX M2, E. coli TEM CTX M9 and S. aureus ATCC 6538 and S. aureus MRSA ATCC 43300 Gram-positive and Gram-negative bacteria strains. Very low MIC values (5 µM) were found for the two resistant strains E.coli TEM CTX M9 and S. aureus MRSA ATCC 43300, in the case of the amide derivative, and for S. aureus MRSA ATCC 43300, in the case of the ester derivative. The interaction of the serine amphiphiles with lipid-model membranes (DPPG and DPPC) was investigated using Langmuir monolayers. A more pronounced effect on the DPPG than on the DPPC monolayer was observed. The effect induced by the surfactants on bacteria membrane was explored by Atomic Force Microscopy. A clear disruption of the bacteria membrane was observed for E. coli TEM CTX M9 upon treatment with (12ser)2CON12, whereas for the S. aureus MRSA few observable changes in cell morphology were found after treatment with either of the two surfactants. The cytotoxicity of the two compounds was assessed by hemolysis assay on human red blood cells (RBC). The compounds were shown to be non-cytotoxic up to 10 µM. Overall, the results reveal a promising potential, in particular of the amide derivative, as antimicrobial agent for two strains of antibiotic resistant bacteria.

Effective cytocompatible nanovectors based on serine-derived gemini surfactants and monoolein for small interfering RNA delivery.

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)2N12, amine derivative; (12Ser)2COO12, ester derivative; and (12Ser)2CON12, 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.

Surface charge tunable catanionic vesicles based on serine-derived surfactants as efficient nanocarriers for the delivery of the anticancer drug doxorubicin.

Self-assembled vesicles composed of amino acid-based cationic/anionic surfactant mixtures show promise as novel effective drug nanocarriers. Here, we report the in vitro performance of vesicles based on cationic (16Ser) and anionic (8-8Ser) serine-based surfactants using a cancer cell model for the delivery of the anticancer drug doxorubicin (DOX). This catanionic mixture yields both negatively (0.20 in the cationic surfactant molar fraction, x16Ser) and positively (x16Ser = 0.58) charged vesicles, hence providing a surface charge tunable system. Low toxicity is confirmed for concentration ranges below 32 µM in both formulations. DOX is successfully encapsulated in the vesicles, resulting in a surface charge switch to negative for the (0.58) system, making both (0.20) and (0.58) DOX-loaded vesicles highly interesting for systemic administration. High uptake by cells was demonstrated using flow cytometry and confocal microscopy. Drug accumulation results in an increase of cell uptake up to 250% and 200% for the (0.20) and (0.58) vesicles, respectively, compared to free DOX and with localizations near the nuclear regions in the cells. The in vitro cytotoxicity studies show that DOX-loaded vesicles induce cell death, confirming the therapeutic potential of the formulations. Furthermore, the efficient accumulation of the drug inside the cell compartments harbors the potential for optimization strategies including phased delivery for prolonged treatment periods or even on-demand release.

New serine-derived gemini surfactants as gene delivery systems.

Gemini surfactants have been extensively used for in vitro gene delivery. Amino acid-derived gemini surfactants combine the special aggregation properties characteristic of the gemini surfactants with high biocompatibility and biodegradability. In this work, novel serine-derived gemini surfactants, differing in alkyl chain lengths and in the linker group bridging the spacer to the headgroups (amine, amide and ester), were evaluated for their ability to mediate gene delivery either per se or in combination with helper lipids. Gemini surfactant-based DNA complexes were characterized in terms of hydrodynamic diameter, surface charge, stability in aqueous buffer and ability to protect DNA. Efficient formulations, able to transfect up to 50% of the cells without causing toxicity, were found at very low surfactant/DNA charge ratios (1/1-2/1). The most efficient complexes presented sizes suitable for intravenous administration and negative surface charge, a feature known to preclude potentially adverse interactions with serum components. This work brings forward a new family of gemini surfactants with great potential as gene delivery systems.

Novel serine-based gemini surfactants as chemical permeation enhancers of local anesthetics: A comprehensive study on structure-activity relationships, molecular dynamics and dermal delivery.

This work aims at studying the efficacy of a series of novel biocompatible, serine-based surfactants as chemical permeation enhancers for two different local anesthetics, tetracaine and ropivacaine, combining an experimental and computational approach. The surfactants consist of gemini molecules structurally related, but with variations in headgroup charge (nonionic vs. cationic) and in the hydrocarbon chain lengths (main and spacer chains). In vitro permeation and molecular dynamics studies combined with cytotoxicity profiles were performed to investigate the permeation of both drugs, probe skin integrity, and rationalize the interactions at molecular level. Results show that these enhancers do not have significant deleterious effects on the skin structure and do not cause relevant changes on cell viability. Permeation across the skin is clearly improved using some of the selected serine-based gemini surfactants, namely the cationic ones with long alkyl chains and shorter spacer. This is noteworthy in the case of ropivacaine hydrochloride, which is not easily administered through the stratum corneum. Molecular dynamics results provide a mechanistic view of the surfactant action on lipid membranes that essentially corroborate the experimental observations. Overall, this study suggests the viability of these serine-based surfactants as suitable and promising delivery agents in pharmaceutical formulations.