Inclusion of a pH-responsive amino acid-based amphiphile in methotrexate-loaded chitosan nanoparticles as a delivery strategy in cancer therapy.

The encapsulation of antitumor drugs in nanosized systems with pH-sensitive behavior is a promising approach that may enhance the success of chemotherapy in many cancers. The nanocarrier dependence on pH might trigger an efficient delivery of the encapsulated drug both in the acidic extracellular environment of tumors and, especially, in the intracellular compartments through disruption of endosomal membrane. In this context, here we reported the preparation of chitosan-based nanoparticles encapsulating methotrexate as a model drug (MTX-CS-NPs), which comprises the incorporation of an amino acid-based amphiphile with pH-responsive properties (77KS) on the ionotropic complexation process. The presence of 77KS clearly gives a pH-sensitive behavior to NPs, which allowed accelerated release of MTX with decreasing pH as well as pH-dependent membrane-lytic activity. This latter performance demonstrates the potential of these NPs to facilitate cytosolic delivery of endocytosed materials. Outstandingly, the cytotoxicity of MTX-loaded CS-NPs was higher than free drug to MCF-7 tumor cells and, to a lesser extent, to HeLa cells. Based on the overall results, MTX-CS-NPs modified with the pH-sensitive surfactant 77KS could be potentially useful as a carrier system for intracellular drug delivery and, thus, a promising targeting anticancer chemotherapeutic agent.

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.

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.

Disturbance of erythrocyte lipid bilayer by amino acid-based surfactants.

In an attempt to increase our knowledge regarding the mechanisms of surfactant membrane interaction, we studied the action of several anionic and cationic amino acid-based surfactants on membrane fluidity using fluorescence anisotropy. Anisotropy measurements demonstrated that almost all of the surfactants studied disturbed the external region of the erythrocyte membrane without affecting the core of the bilayer. How the physico-chemical properties and structure of these compounds affect dynamics of the lipid bilayer is discussed in detail.

In vitro studies of the hemolytic activity of microemulsions in human erythrocytes.

Hemolytic activity in human erythrocytes as alternative to in vivo testing was used as a potential screening method to evaluate irritant potential of microemulsions for possible application in pharmaceutical and cosmetic formulations. Microemulsions were prepared by mixing surfactants and oil and slowly titrating the mixtures with aliquots of phosphate buffer saline or water. All microemulsions were characterized by dynamic light scattering to determine both the mean droplet size and droplet distribution. Microemulsion droplet size decreased as aqueous component increased. No differences in droplet size were observed between formulations containing phosphate buffered saline or water. The hemolytic activity was measured photometrically by the RBC assay, based in the cell membrane lysis, to estimate the potential irritation of both surfactants and microemulsions selected with water or PBS as aqueous component. The most hemolytic microemulsions corresponded to those containing the surfactant Labrasol, with or without butyl lactate, and no differences were found between the hemolytic activity between these components and microemulsions containing them. The highest hemolytic activity of microemulsions in this study may be attributed to the excipient used in the formulations. We should avoid the use of high amounts of Labrasol and butyl lactate in microemulsions because they may be potential irritants.