M. Rosa Infante

Cationic surfactants from lysine: Synthesis, micellization and biological evaluation

Biocompatible cationic surfactants from the amino acid lysine (hydrochloride salts of N(epsilon)-lauroyl lysine methyl ester, N(epsilon)-myristoyl lysine methyl ester and N(epsilon)-palmitoyl lysine methyl ester) have been prepared in high yields by lysine acylation in epsilon position with three natural saturated fatty acids. The micellization process of these surfactants has been studied using the PGSE-NMR technique. The compounds were tested as antimicrobial agents against Gram-positive and Gram-negative bacteria. The surfactants show moderate antimicrobial activity against the Gram-positive bacteria but Gram-negative bacteria are resistant to these surfactants in the concentration range tested. The haemolytic activity is considerably lower than those reported for other cationic N(alpha)-acyl amino acid analogues. The acute toxicity against Daphnia magna and biodegradability was studied. The toxicity is clearly lower than that reported for conventional cationic surfactants from quaternary ammonium and the three surfactants from lysine can be classified as ready biodegradable surfactants.

Low Potential Ocular Irritation of Arginine-Based Gemini Surfactants and Their Mixtures with Nonionic and Zwitterionic Surfactants

AbstractPurpose. The aim of this study was to find new biocompatible surfactants and mixtures with low ocular irritant action for application in pharmaceutical formulations and to establish a relationship between their structure and their potential ocular irritant activity. Methods. An alternative method to the Draize in vivo test, based on the adverse effects of surfactants on the cytoplasmic membrane of red blood cell, was used to evaluate the potential ocular irritation of the surfactants. Results. It was found that the hemolytic activity of arginine-based gemini surfactants increased with the aliphatic alkyl chain lengths of the hydrophobic tail. The addition of the surfactant with an alkyl chain length of 10 carbon atoms to cocoamidopropilbetaina (TB), decylglucoside (APG), and Nα-lauroyl-arginine ethyl ester (LAE) increases the hemolytic activity moderately for the mixtures with TB and LAE (1.1- and 1.5-fold, respectively) and strongly for APG (fivefold). Conclusions. The new arginine-based gemini surfactants constitute a suitable alternative to commercial surfactants because of their natural origins, which make them biocompatible and renewable products. Based on their hemolytic activity as an alternative to the Draize test, these new arginine-based gemini surfactants and their mixtures can be classified as mild irritants. This fact constitutes an advantage, especially for pharmaceutical and cosmetic applications.

Comparative sensitivity of tumor and non-tumor cell lines as a 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. IC(50)-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.

PROPERTIES OF LIPOAMINO ACIDS INCORPORATED INTO MEMBRANE BILAYERS

Several lipoamino acids were synthesized in which palmitic acid was coupled with the alpha-amino group of an amino acid. These lipoamino acids were tested for their inhibitory action against Sendai virus fusion to liposomes composed of egg phosphatidylethanolamine and 5 mol% of the ganglioside GD1a. A commonly employed viral fusion assay based on the dilution of the fluorescent probe octadecylrhodamine (R18) exhibited an additional complication in the presence of Nalpha-palmitoyl tryptophan (palm-Trp). At higher mol fraction of palm-Trp it was observed that there was an increase in R18 quenching. Studies on the dependence of the emission wavelength of palm-Trp on excitation wavelength demonstrated that the presence of R18 alters the environment of the indole. The results illustrate one of the complexities of viral fusion assays using the R18 probe. Despite this complication it was possible to demonstrate that several of the lipoamino acids are effective at inhibiting the fusion of Sendai virus to liposomes as measured by the R18 assay. One of the most effective inhibitors of this process is palm-Trp which, at a concentration of 4 mol% in liposomes, markedly reduces the apparent rate of fusion. At pH 5.0 this amphiphile is also an inhibitor of Sendai virus fusion, indicating that the ionization of the carboxyl group of this amphiphile is not required for its antiviral activity. The inhibitory action of palm-Trp against Sendai virus was confirmed by demonstrating inhibition of Sendai-mediated cytopathic effects studied in tissue culture. A property associated with antiviral activity is the ability of amphiphiles to raise the bilayer to hexagonal phase transition temperature of dielaidoyl phosphatidylethanolamine. All of these lipoamino acids were found to possess this property, but a quantitative relationship with inhibition of viral fusion was not found.

PEGylated and poloxamer-modified chitosan nanoparticles incorporating a lysine-based surfactant for pH-triggered doxorubicin release

The growing demand for efficient chemotherapy in many cancers requires novel approaches in target-delivery technologies. Nanomaterials with pH-responsive behavior appear to have potential ability to selectively release the encapsulated molecules by sensing the acidic tumor microenvironment or the low pH found in endosomes. Likewise, polyethylene glycol (PEG)- and poloxamer-modified nanocarriers have been gaining attention regarding their potential to improve the effectiveness of cancer therapy. In this context, DOX-loaded pH-responsive nanoparticles (NPs) modified with PEG or poloxamer were prepared and the effects of these modifiers were evaluated on the overall characteristics of these nanostructures. Chitosan and tripolyphosphate were selected to form NPs by the interaction of oppositely charged compounds. A pH-sensitive lysine-based amphiphile (77KS) was used as a bioactive adjuvant. The strong dependence of 77KS ionization with pH makes this compound an interesting candidate to be used for the design of pH-sensitive devices. The physicochemical characterization of all NPs has been performed, and it was shown that the presence of 77KS clearly promotes a pH-triggered DOX release. Accelerated and continuous release patterns of DOX from CS-NPs under acidic conditions were observed regardless of the presence of PEG or poloxamer. Moreover, photodegradation studies have indicated that the lyophilization of NPs improved DOX stability under UVA radiation. Finally, cytotoxicity experiments have shown the ability of DOX-loaded CS-NPs to kill HeLa tumor cells. Hence, the overall results suggest that these pH-responsive CS-NPs are highly potent delivery systems to target tumor and intracellular environments, rendering them promising DOX carrier systems for cancer therapy.

Novel Biocompatible DNA Gel Particles

Surfactants with the cationic functionality based on an amino acid structure have been used to prepare novel biocompatible devices for the controlled encapsulation and release of DNA. We report here the formation of DNA gel particles mixing DNA (either single- (ssDNA) or double-stranded (dsDNA)) with two different single-chain amino acid-based surfactants: arginine-N-lauroyl amide dihydrochloride (ALA) and N(alpha)-lauroyl-arginine-methyl ester hydrochloride (LAM). The degree of DNA entrapment, the swelling/deswelling behavior, and the DNA release kinetics have been studied as a function of both the number of charges in the polar head of the amino acid-based surfactant and the secondary structure of the nucleic acid. Analysis of the data indicates a stronger interaction of ALA with DNA, compared with LAM, mainly attributed to the double charge carried by the former surfactant compared to the singly charged headgroup of the latter species. The stronger interaction with amphiphiles for ssDNA compared with dsDNA suggests the important role of hydrophobic interactions in DNA. Data on the microstructure of the complexes obtained from small-angle X-ray scattering (SAXS) of the particles strongly suggests a hexagonal packing. It was found that, the shorter the lattice parameter, the stronger the surfactant-DNA interaction and the slower the DNA release kinetics. Complexation and neutralization of DNA on the DNA gel particles was confirmed by agarose gel electrophoresis measurements.

Biological properties of arginine-based glycerolipidic cationic surfactants

Amino acid-based surfactants have attracted much interest as environmentally friendly surfactants because of their biodegradability, low aquatic toxicity, and low haemolytic activity. Our group has recently developed a new family of arginine-based glycerolipidic surfactants: 1-acyl-3-O-(L-arginyl)-rac-glycerol·2HCl (X0R) and 1,2-diacyl-3-O-(L-arginyl)-rac-glycerol·2HCl (XXR) with alkyl chain lengths in the range of C8–C14. In this paper we study the biodegradability and aquatic toxicity of these recently developed arginine-based glycerolipidic cationic surfactants and compare these to conventional cationic surfactants.

Sequestration of bacterial lipopolysaccharide by bis(Args) gemini compounds.

Gemini compounds of the type N(alpha),N(omega)-bis(N(alpha)-lauroyl arginine)alpha,omega-alkylenediamides or bis(Args) bind bacterial lipopolysaccharide and neutralize endotoxic activity in in vitro tumor necrosis factor-alpha and nitric oxide release assays. Sequestration of lipopolysaccharide results in protection in a murine model of endotoxemia. However, the bis(Args) compounds are cytotoxic by virtue of being highly membrane-active. The development of less surface-active analogues may yield potentially therapeutically useful compounds for the treatment of Gram-negative sepsis.

Nanoparticles incorporating pH-responsive surfactants as a viable approach to improve the intracellular drug delivery.

The pH-responsive delivery systems have brought new advances in the field of functional nanodevices and might allow more accurate and controllable delivery of specific cargoes, which is expected to result in promising applications in different clinical therapies. Here we describe a family of chitosan-TPP (tripolyphosphate) nanoparticles (NPs) for intracellular drug delivery, which were designed using two pH-sensitive amino acid-based surfactants from the family N(α),N(ε)-dioctanoyl lysine as bioactive compounds. Low and medium molecular weight chitosan (LMW-CS and MMW-CS, respectively) were used for NP preparation, and it was observed that the size distribution for NPs with LMW-CS were smaller (~168 nm) than that for NPs prepared with MMW-CS (~310 nm). Hemolysis assay demonstrated the pH-dependent biomembrane disruptional capability of the constructed NPs. The nanostructures incorporating the surfactants cause negligible membrane permeabilization at pH7.4. However, at acidic pH, prevailing in endosomes, membrane-destabilizing activity in an erythrocyte lysis assay became evident. When pH decreased to 6.6 and 5.4, hemolytic capability of chitosan NPs increased along with the raise of concentration. Furthermore, studies with cell culture showed that these pH-responsive NPs displayed low cytotoxic effects against 3T3 fibroblasts. The influence of chitosan molecular weight, chitosan to TPP weight ratio, nanoparticle size and nature of the surfactant counterion on the membrane-disruptive properties of nanoparticles was discussed in detail. Altogether, the results achieved here showed that by inserting the lysine-based amphiphiles into chitosan NPs, pH-sensitive membranolytic and potentially endosomolytic nanocarriers were developed, which, therefore, demonstrated ideal feasibility for intracellular drug delivery.

Surface activity and aggregation of pristine and hydrophobically modified inulin

In search of more effective and lower impact surfactants, hydrophobic modification of polymers has emerged as a promising strategy for the substitution of low molecular weight surfactants. Inulin, a polysaccharide obtained from chicory roots, has links with the sustainable chemistry model. Furthermore, hydrophobically modified inulins (HMI) can be synthesized in aqueous media. This article reports on the surface activity and aggregation of several compounds with differences in hydrophobic alkyl length and backbone linkage. HMI significantly reduces (down to 30 mN m−1) water surface tension while inulin does not. Both inulins and HMI are shown to aggregate hierarchically at three levels, namely aggregates ( 500 nm). The smaller aggregates, which in the case of inulins can be classified as monomers and in the case of HMI as micelles, have dimensions below 15 nm. The second level of aggregation corresponds to loose flocks with dimensions up to 100 nm and the third level corresponds to the precipitate constituted by aggregation of the flocks. Temperature and basic conditions affect the flock formation and kinetics without a strong influence on the smaller aggregates structure and size. Long term standing of both HMI and inulin solutions eventually precipitates. This precipitate may occupy a large volume but corresponds to a small amount of the solute. In the case of inulins, the precipitate has been found to have a degree of crystallinity that diminishes with increasing temperature.