Assessment of the potential skin irritation of lysine-derivative anionic surfactants using mouse fibroblast and human keratinocytes as an alternative to animal testing

Purpose. The aim of this study was to identify new surfactants with low skin irritant properties for use in pharmaceutical and cosmetic formulations, employing cell culture as an alternative method to in vivo testing. In addition, we sought to establish whether potential cytotoxic properties were related to the size of the counterions bound to the surfactants. Methods. Cytotoxicity was assessed in the mouse fibroblast cell line 3T6, and the human keratinocyte cell line NCTC 2544, using the MTT assay and uptake of the vital dye neutral red 24 h after dosing (NRU). Results. Lysine-derivative surfactants showed higher IC50s than did commercial anionic irritant compounds such as sodium dodecyl sulphate, proving to be no more harmful than amphoteric betaines. The aggressiveness of the surfactants depended upon the size of their constituent counterions: surfactants associated with lighter counterions showed a proportionally higher aggressivity than those with heavier ones. Conclusions. Synthetic lysine-derivative anionic surfactants are less irritant than commercial surfactants such as sodium dodecyl sulphate and Hexadecyltrimethylammonium bromide and are similar to Betaines. These surfactants may offer promising applications in pharmaceutical and cosmetic preparations, representing a potential alternative to commercial anionic surfactants as a result of their low irritancy potential.

The role of counterions in the membrane-disruptive properties of pH-sensitive lysine-based surfactants

Surfactants are among the most versatile and widely used excipients in pharmaceuticals. This versatility, together with their pH-responsive membrane-disruptive activity and low toxicity, could also enable their potential application in drug delivery systems. Five anionic lysine-based surfactants which differ in the nature of their counterion were studied. Their capacity to disrupt the cell membrane was examined under a range of pH values, concentrations and incubation times, using a standard hemolysis assay as a model for endosomal membranes. The surfactants showed pH-sensitive hemolytic activity and improved kinetics at the endosomal pH range. Low concentrations resulted in negligible hemolysis at physiological pH and high membrane lytic activity at pH 5.4, which is in the range characteristic of late endosomes. With increasing concentration, the surfactants showed an enhanced capacity to lyse cell membranes, and also caused significant membrane disruption at physiological pH. This observation indicates that, at high concentrations, surfactant behavior is independent of pH. The mechanism of surfactant-mediated membrane destabilization was addressed, and scanning electron microscopy studies were also performed to evaluate the effects of the compounds on erythrocyte morphology as a function of pH. The in vitro cytotoxicity of the surfactants was assessed by MTT and NRU assays with the 3T3 cell line. The influence of different types of counterion on hemolytic activity and the potential applications of these surfactants in drug delivery are discussed. The possibility of using pH-sensitive surfactants for endosome disruption could hold great promise for intracellular drug delivery systems in future therapeutic applications.

Estudi de la desadsorció del pirè amb diferents surfactants en tres sòls artificials

Un dels problemes associats a la remeiació de contaminants hidròfobs és la seva poca disponibilitat. Es considera que un contaminant està disponible quan roman a la fase líquida del medi, ja sigui solubilitzat o en forma d’emulsió. Els surfactants són substàncies anfifíliques que promouen la transferència de compostos hidròfobs de la fase sòlida a la líquida. En aquest estudi s’escull el pirè com a representant dels hidrocarburs aromàtics policíclics i tres surfactants no iònics: un àmpliament citat a la bibliografia científica (Tween 80) i dos comercials (Gold Crew, BS-400). L’estudi es fa amb tres mescles d’argila i sorra amb diferents proporcions. La concentració micel·lar crítica (CMC) s’assoleix abans en sòls amb poc contingut en argila. L’eficiència dels surfactants està estretament relacionada amb la proporció d’argila i sorra. A concentracions molt per sobre de la CMC no s’observa una relació entre l’eficiència i la quantitat d’argila. El Tween 80 ha donat millors resultats que el Gold Crew i el BS-400, sense que aquest darrer no hagi presentat desadsorció de pirè.

Comparative sensitivity of tumor and non-tumor cell lines as reliable approach for in vitro cytotoxicity screening of lysine-based surfactants with potential pharmaceutical applications

Surfactants are used as additives in topical pharmaceuticals and drug delivery systems. The biocompatibility of amino acid-based surfactants makes them highly suitable for use in these fields, but tests are needed to evaluate their potential toxicity. Here we addressed the sensitivity of tumor (HeLa, MCF-7) and non-tumor (3T3, 3T6, HaCaT, NCTC 2544) cell lines to the toxic effects of lysine-based surfactants by means of two in vitro endpoints (MTT and NRU). This comparative assay may serve as a reliable approach for predictive toxicity screening of chemicals prior to pharmaceutical applications. After 24-h of cell exposure to surfactants, differing toxic responses were observed. NCTC 2544 and 3T6 cell lines were the most sensitive, while both tumor cells and 3T3 fibroblasts were more resistant to the cytotoxic effects of surfactants. IC50-values revealed that cytotoxicity was detected earlier by MTT assay than by NRU assay, regardless of the compound or cell line. The overall results showed that surfactants with organic counterions were less cytotoxic than those with inorganic counterions. Our findings highlight the relevance of the correct choice and combination of cell lines and bioassays in toxicity studies for a safe and reliable screen of chemicals with potential interest in pharmaceutical industry.

Membrane-destabilizing activity of pH-responsive cationic lysine-based surfactants: role of charge position and alkyl chain length

Many strategies for treating diseases require the delivery of drugs into the cell cytoplasm following internalization within endosomal vesicles. Thus, compounds triggered by low pH to disrupt membranes and release endosomal contents into the cytosol are of particular interest. Here, we report novel cationic lysine-based surfactants (hydrochloride salts of N¿- and N¿-acyl lysine methyl ester) that differ in the position of the positive charge and the length of the alkyl chain. Amino acid-based surfactants could be promising novel biomaterials in drug delivery systems, given their biocompatible properties and low cytotoxic potential. We examined their ability to disrupt the cell membrane in a range of pH values, concentrations and incubation times, using a standard hemolysis assay as a model of endosomal membranes. Furthermore, we addressed the mechanism of surfactant-mediated membrane destabilization, including the effects of each surfactant on erythrocyte morphology as a function of pH. We found that only surfactants with the positive charge on the ¿-amino group of lysine showed pH-sensitive hemolytic activity and improved kinetics within the endosomal pH range, indicating that the positive charge position is critical for pH-responsive behavior. Moreover, our results showed that an increase in the alkyl chain length from 14 to 16 carbon atoms was associated with a lower ability to disrupt cell membranes. Knowledge on modulating surfactant-lipid bilayer interactions may help us to develop more efficient biocompatible amino acid-based drug delivery devices.

pH Sensitive surfactants from lysine: assessment of their cytotoxicity and environmental behavior

The toxicity and environmental behavior of new pH-sensitive surfactants from lysine are presented. Three different chemical structures are studied: surfactants with one amino acid and one alkyl chain, surfactants with two amino acids on the polar head and one alkyl chain, and gemini surfactants. The pH sensitivity of these compounds can be tuned by modifying their chemical structures. Cytotoxicity has been evaluated using erythrocytes and fibroblast cells. The toxic effects against these cells depend on the hydrophobicity of the molecules as well as their cationic charge density. The effect of hydrophobicity and cationic charge density on toxicity is different for each type of cells. For erythrocytes, the toxicity increases as hydrophobicity and charge density increases. Nevertheless, for fibroblasts cationic charge density affects cytotoxicity in the opposite way: the higher charge density, the lower the toxicity. The effect of the pH on hemolysis has been evaluated in detail. The aquatic toxicity was established using Daphnia magna. All surfactants yielded EC50 values considerably higher than that reported for cationic surfactants based on quaternary ammonium groups. Finally, their biodegradability was evaluated using the CO2 headspace test (ISO 14593). These lysine derivatives showed high levels of biodegradation under aerobic conditions and can be classified as"readily biodegradable compounds".

pH Sensitive surfactants from lysine: assessment of their cytotoxicity and environmental behavior

The toxicity and environmental behavior of new pH-sensitive surfactants from lysine are presented. Three different chemical structures are studied: surfactants with one amino acid and one alkyl chain, surfactants with two amino acids on the polar head and one alkyl chain, and gemini surfactants. The pH sensitivity of these compounds can be tuned by modifying their chemical structures. Cytotoxicity has been evaluated using erythrocytes and fibroblast cells. The toxic effects against these cells depend on the hydrophobicity of the molecules as well as their cationic charge density. The effect of hydrophobicity and cationic charge density on toxicity is different for each type of cells. For erythrocytes, the toxicity increases as hydrophobicity and charge density increases. Nevertheless, for fibroblasts cationic charge density affects cytotoxicity in the opposite way: the higher charge density, the lower the toxicity. The effect of the pH on hemolysis has been evaluated in detail. The aquatic toxicity was established using Daphnia magna. All surfactants yielded EC50 values considerably higher than that reported for cationic surfactants based on quaternary ammonium groups. Finally, their biodegradability was evaluated using the CO2 headspace test (ISO 14593). These lysine derivatives showed high levels of biodegradation under aerobic conditions and can be classified as"readily biodegradable compounds".

Phospholipid bilayer perturbing-properties underlying lysis induced by pH-sensitive cationic lysine-based surfactants in biomembranes

Many strategies for treating diseases require the delivery of drugs into the cell cytoplasm following internalization within endosomal vesicles. Thus, compounds triggered by low pH to disrupt membranes and release endosomal contents into the cytosol are of particular interest. Here, we report novel cationic lysine-based surfactants (hydrochloride salts of Nε- and Nα-acyl lysine methyl ester) that differ in the position of the positive charge and the length of the alkyl chain. Amino acid-based surfactants could be promising novel biomaterials in drug delivery systems, given their biocompatible properties and low cytotoxic potential. We examined their ability to disrupt the cell membrane in a range of pH values, concentrations and incubation times, using a standard hemolysis assay as a model of endosomal membranes. Furthermore, we addressed the mechanism of surfactant-mediated membrane destabilization, including the effects of each surfactant on erythrocyte morphology as a function of pH. We found that only surfactants with the positive charge on the α-amino group of lysine showed pH-sensitive hemolytic activity and improved kinetics within the endosomal pH range, indicating that the positive charge position is critical for pH-responsive behavior. Moreover, our results showed that an increase in the alkyl chain length from 14 to 16 carbon atoms was associated with a lower ability to disrupt cell membranes. Knowledge on modulating surfactant-lipid bilayer interactions may help us to develop more efficient biocompatible amino acid-based drug delivery devices.

New cationic nanovesicular systems containing lysine-based surfactants for topical administration: Toxicity assessment using representative skin cell lines

Many strategies for treating diseases require the delivery of drugs into the cell cytoplasm following internalization within endosomal vesicles. Thus, compounds triggered by low pH to disrupt membranes and release endosomal contents into the cytosol are of particular interest. Cationic nanovesicles have attracted considerable interest as effective carriers to improve the delivery of biologically active molecules into and through the skin. In this study, lipid-based nanovesicles containing three different cationic lysine-based surfactants were designed for topical administration. We used representative skin cell lines and in vitro assays to assess whether the cationic compounds modulate the toxic responses of these nanocarriers. The nanovesicles were characterized in both water and cell culture medium. In general, significant agglomeration occurred after 24 h incubation under cell culture conditions. We found different cytotoxic responses among the formulations, which depended on the surfactant,cell line (3T3, HaCaT, and THP-1) and endpoint assayed (MTT, NRU, and LDH). Moreover, no potential phototoxicity was detected in fibroblast or keratinocyte cells, whereas only a slight inflammatory response was induced, as detected by IL-1a and IL-8 production in HaCaT and THP-1 cell lines, respectively. A key finding of our research was that the cationic charge position and the alkyl chain length of the surfactants determine the nanovesicles resulting toxicity. The charge on the a-amino group of lysine increased the depletion of cell metabolic activity, as determined by the MTT assay, while a higher hydrophobicity tends to enhance the toxic responses of the nanovesicles. The insights provided here using different cell lines and assays offer a comprehensive toxicological evaluation of this group of new nanomaterials.

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

Understanding nanomaterial interactions within cells is of increasing importance for assessing their toxicity and cellular transport. Here, we developed nanovesicles containing bioactive cationic lysine-based amphiphiles, and assessed whether these cationic compounds increase the likelihood of intracellular delivery and modulate toxicity. We found different cytotoxic responses 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 internalized 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 behavior after cell uptake of the nanomaterial. The insights into some toxicity mechanisms of these new nanomaterials contribute to reducing the uncertainty surrounding their potential health hazards.

Preparació de materials híbrids orgànico-inorgànics a partir de sals d'imidazoli

Estudi elaborat a partir d’una estada a l’ Ecole Nationale Supérieure de Chimie de Montpellier, França, durant 2006. S’han sintetitzat materials híbrids orgànico-inorgànics mitjançant el procés sol-gel i altres estratègies sintètiques. En alguns casos, s’ha intentat estructurar aquests materials, ja sigui per autoestructuració o per mitjà de tensioactius. Com a catalitzadors de les reaccions d'hidròlisi i policondensació s’han utilitzat àcids, bases i fluorurs. Els materials obtinguts s’han caracteritzat mitjançant diferents tècniques: BET (Brunauer-Emmett-Teller), TEM (microscopia electrònica de transmissió), SEM (microscòpia electrònica de rastreig), raigs X en pols , IR i RMN (ressonància magnètica nuclear) en estat sòlid. Amb aquests materials es pretén preparar catalitzadors heterogenis de Pd per reaccions d’acoblament creuat, i de Ru per reaccions de metàtesi. També s’han sintetitzat sals d'imidazoli amb cadenes hidrocarbonades llargues amb l'objectiu de preparar gels de sílice amb aquestes molècules atrapades dins la matriu inorgànica. Aquests materials s’utilitzaran com a organocatalitzadors i també es prepararan els corresponents catalitzadors de Pd per reaccions de Heck, Suzuki i Sonogashira. Les sals d’imidazoli s’han utilitzat com a tensioactius en la preparació de gels de sílice estructurats. Aquestes molècules han resultat ser cristalls líquids i s’han caracteritzar mitjançant DSC (differential scanning calorimetry), microscopia òptica i raigs X.

Preparació i caracterització de materials mesoporosos en sistemes tensioactius

Estudi elaborat a partir d’una estada a la Universitat Nacional de Yokohama des de maig fins a mitjans de juny del 2006. S'ha estudiat el comportament fàssic i la preparació de sílica mesoporosa pels nous tensioactius fluorats d'estructura C8F17SO2(C3H7)N(C2H4O)nH (abreujat C8F17(EO)n. El tensioactiu C8F17(EO)n forma micel•les allargades i cristalls líquids en aigua, i per tant pot ser adequat per a la preparació de materials mesoporosos. Sílica mesoestructurada es va preparar pel mètode de precipitació per autoagregació cooperativa. Un estudi sistemàtic es va realitzar, investigant la influència de les concentracions de tensioactiu i precursor (TEOS), l’efecte del pH i de la longitud de cadena de poliòxid d’etilè. Els materials es van caracteritzar per raigs X a angle petit (SAXS), sorció de nitrògen i TEM. Els materials obtinguts presenten diàmetres de por petits i parets de por gruixudes. A més, aquests materials posseeixen altes superfícies específiques, que s’han obtingut emprant concentracions de tensioactiu petites, produint parets de por robustes sense microporositat significativa. La superfície específica es manté durant el procés de calcinació, malgrat un petit encongiment degut a l’entrecreuament de la sílica. Els materials de sílica obtinguts han mostrat ser significativament més robustos que altres materials similars descrits a la bibliografia, com la sílica MCM-41.

Estudi del comportament de materials nanoestructurats en el tractament d'aigües contaminades amb arsènic

El SPION (Super Paramagnetic Iron Oxide Nanoparticles) ha estat estudiat com un nou adsorbent per eliminar l’arsènic d’aigües contaminades. Les condicions òptimes de treball es van assolir per un pH de 3,6 i per concentracions inferiors als 100ppm. No es van trobar interferències significatives produïdes pels cations Cu, Ni i Zn en l’adsorció de l’As, sent el fosfat l’anió que més interfereix. Una esponja de cel·lulosa s’ha utilitzat com a suport del SPION, per disminuir les agregacions de les nanopartícules en suspensió i per proporcionar una material adequat per l’adsorció en continu, experiment amb columnes. Així, es va obtenir un augment de la capacitat d’adsorció del SPION per l’As(V), mentre que per l’As(III) continua sent baixa, per tant s’augmenta la selectivitat per l’As(V). Les interferències aniòniques afecten d’igual manera a l’adsorció de l’As(III) i l’As(V) quan l’adsorció és en continu o en discontinu. Els cations metàl·lics no interfereixen en l’adsorció de l’arsènic, a excepció del coure que és adsorbit i porta a la disminució de l’adsorció d’arsènic.

Experimental and computational study on the synthesis and characterization of self-assembling meso/macroporous materials in highly concentrated emulsions

Report for the scientific sojourn carried out at Massachusetts General Hospital Cancer Center-Harvard Medical School, Estats Units, from 2010 to 2011. The project aims to study the aggregation behavior of amphiphilic molecules in the continuous phase of highly concentrated emulsions, which can be used as templates for the synthesis of meso/macroporous materials. At this stage of the project, we have investigated the self-assembly of diblock and triblock surfactants under the effect of a confined geometry being surrounded by the droplets of the dispersed phase. These droplets limit the growth of the aggregates, deeply modify their orientation and hence alter their spatial arrangement as compared to the self-assembly taking place far enough from any boundary surface, that is in the bulk. By performing Monte Carlo simulations, we have showed that the interface between the dispersed and continuous phases as well as its shape has a significant impact on the structural order of the resulting aggregates and hence on the potential applications of highly concentrated emulsions as reaction media, drug delivery systems, or templates for meso/macroporous materials. Due to the combined effect of symmetry breaking and morphological frustration, very intriguing structures, such as square columnar liquid crystals, twisted X-shaped aggregates, and helical phases of cylindrical aggregates, never observed in the bulk for the same model surfactant, have been found. The presence of other more conventional structures, such as micelles and cubic and hexagonal liquid crystals, formed at low and high amphiphilic concentrations, respectively, further enhance the interest on this already rich aggregation behavior.

Counter-ion effect on surfactant-DNA gel particles as controlled DNA delivery systems

The ability to entrap drugs within vehicles and subsequently release them has led to new treatments for a number of diseases. Based on an associative phase separation and interfacial diffusion approach, we developed a way to prepare DNA gel particles without adding any kind of cross-linker or organic solvent. Among the various agents studied, cationic surfactants offered particularly efficient control for encapsulation and DNA release from these DNA gel particles. The driving force for this strong association is the electrostatic interaction between the two components, as induced by the entropic increase due to the release of the respective counter-ions. However, little is known about the influence of the respective counter-ions on this surfactant-DNA interaction. Here we examined the effect of different counter-ions on the formation and properties of the DNA gel particles by mixing DNA (either single- (ssDNA) or double-stranded (dsDNA)) with the single chain surfactant dodecyltrimethylammonium (DTA). In particular, we used as counter-ions of this surfactant the hydrogen sulfate and trifluoromethane sulfonate anions and the two halides, chloride and bromide. Effects on the morphology of the particles obtained, the encapsulation of DNA and its release, as well as the haemocompatibility of these particles, are presented, using the counter-ion structure and the DNA conformation as controlling parameters. Analysis of the data indicates that the degree of counter-ion dissociation from the surfactant micelles and the polar/hydrophobic character of the counter-ion are important parameters in the final properties of the particles. The stronger interaction with amphiphiles for ssDNA than for dsDNA suggests the important role of hydrophobic interactions in DNA.