M. Carmen Morán

Cationic agents for DNA compaction

Fluorescence microscopy was used to investigate the conformational changes of individual T4 DNA molecules induced by different compacting agents, namely the cationic surfactants, cetyltrimethylammonium bromide (CTAB) and chloride (CTAC), iron(III), lysozyme, and protamine sulfate. A protocol for establishing size estimates is suggested to obtain reproducible results. Observations show that in the presence of lysozyme and protamine sulfate, DNA molecules exhibit a conformational change from an elongated coil structure to compact globules, usually interpreted as a first-order transition. The maximum degree of compaction that is attained when iron(III) or CTAB (CTAC) are used as compacting agents is considerably smaller, and intermediate structures (less elongated coils) are visible even for high concentrations of these agents. Dynamic light scattering experiments were carried out, for some of the systems, to assess the reliability of size estimates from fluorescence microscopy.

DNA gel particles

This review covers recent developments on the topic of DNA gel particles formed in water–water emulsion-type interfaces. A general understanding of interactions between DNA and oppositely charged agents has given us a basis for developing novel DNA-based materials, including gel particles. The association strength, which is tuned by varying the chemical structure of the cationic cosolute, determines the spatial homogeneity of the gelation process, creating DNA reservoir devices and DNA matrix devices that can be designed to release DNA (either single- (ssDNA) or double-stranded (dsDNA)). Besides an introduction concerning general aspects about DNA–cationic complexes and the formation of gels in water–water emulsion-type interfaces and some conclusions, the review contains sections reviewing the preparation of DNA gel particles using 1) surfactants, 2) polysaccharides and 3) proteins. The particle morphology, swelling/dissolution behaviour, degree of DNA entrapment and DNA release responses as a function of the nature of the cationic agent used are discussed. Finally, current directions on the preparation of DNA gel particles, including the decrease of size and the improvement of the biocompatibility of these systems have been reviewed.

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.

Mixed Protein Carriers for Modulating DNA Release

Aqueous mixtures of oppositely charged polyelectrolytes undergo associative phase separation, resulting in coacervation, gelation, or precipitation. This phenomenon has been exploited in forming DNA gel particles by interfacial diffusion. We report here the formation of DNA gel particles by mixing solutions of double-stranded DNA with aqueous solutions containing two cationic proteins, lysozyme and protamine sulfate. The effect of the lysozyme/protamine ratio on the degree of DNA entrapment, surface morphology, swelling-deswelling behavior, and kinetics of DNA release has been investigated. By mixing the two proteins, we obtain particles that display higher loading efficiency and loading capacity values, in comparison to those obtained in single-protein systems. Examination of the release profiles has shown that in mixed protein particles, complex, dual-stage release kinetics is obtained. The overall release profile is dependent on the lysozyme/protamine ratio. The obtained profiles, or segments of them, are accuratelly fitted using the zero-order and first-order models, and the Weibull function. Fluorescence microscopy studies have suggested that the formation of these particles is associated with the conservation of the secondary structure of DNA. This study presents a new platform for controlled release of DNA from DNA gel particles formed by interfacial diffusion.

Controlling the Morphology in DNA Condensation and Precipitation

This work addresses the influence of solution inhomogeneity on conformation, aggregation, and coil/globule and bundle/single chain coexistence of T4 DNA molecules. The inhomogeneity is induced by mixing two solutions containing, respectively, protamine and DNA, with different relative concentrations, but aiming at producing the same final concentrations. The study was conducted by means of fluorescence microscopy (FM), complemented with scanning electron microscopy (SEM). It is shown that the degree of precipitation, the structures formed, and the relative population of compacted and unfolded structures are highly dependent on the method of preparation of the mixtures that contain the DNA/protamine complexes. Most of the structures reported in the literature, that is, overcharged/undercharged globules, toroids, chains internally segregated, and bundles composed of several chains were observed in our different mixtures of fixed final concentration.

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 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

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 24h 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-1α 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 α-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.

DNA gel particles: An overview

A general understanding of interactions between DNA and oppositely charged compounds forms the basis for developing novel DNA-based materials, including gel particles. The association strength, which is altered by varying the chemical structure of the cationic cosolute, determines the spatial homogeneity of the gelation process, creating DNA reservoir devices and DNA matrix devices that can be designed to release either single- (ssDNA) or double-stranded (dsDNA) DNA. This review covers recent developments on the topic of DNA gel particles formed in water-water emulsion-type interfaces. The degree of DNA entrapment, particle morphology, swelling/dissolution behavior and DNA release responses are discussed as functions of the nature of the cationic agent used. On the basis of designing DNA gel particles for therapeutic purposes, recent studies on the determination of the surface hydrophobicity and the hemolytic and the cytotoxic assessments of the obtained DNA gel particles have been also reported.

The nanostructure of surfactant-DNA complexes with different arrangements.

The nanostructure of DNA with different cationic surfactant has been studied in order to elucidate the detailed arrangement concerning the position of DNA and surfactant domains in the complexes. Also, the orientation of the DNA cylinders in the thin films of the complexes was investigated. Attention was directed on the preparation methods of the complexes and to how the different surfactant structure affects the compaction of the DNA. The cationic surfactant-DNA complexes were investigated by X-ray scattering, polarized light microscopy and elemental microanalysis. It was observed that the molecular organization of the complexes between DNA and cationic surfactant corresponds to a hexagonal structure with different packing arrangements. The nanostructure of the complexes depends on the hydrophobic/hydrophilic balance of the cationic surfactant. In particular the use of arginine derived surfactants, with a large polar head group able to interact not only by electrostatics but also by hydrogen bonding, allows for the formation of more compact structures. The results suggest that the smaller the lattice parameter the more compact and stable is the complex implying slower DNA release.

Sustainable DNA Release from Chitosan/Protein Based-DNA Gel Particles

Chitosan lactate (CL) alone and in combination with protamine sulfate (PS) was used as an intrinsic biocompatible carrier to form DNA gel particles by interfacial diffusion. Protamine sulfate is highly positively charged, arginine-rich protein, which has been previosly used in the formation of mixed carriers for modulating DNA release. In view of the promising properties of oligosaccharides and the well-known cell-penetrating and nuclear localization capabilities of protamines, we presume that both structures could play a critical role in DNA delivery. The purpose of this study was to evaluate the capability of water-soluble, low molecular weight chitosan lactate to form DNA gel particles alone (binary system) and in combination with the protein protamine sulfate (ternary system). The particles were characterized with respect to the degree of DNA entrapment, the swelling and dissolution behavior, the secondary structure of DNA in the particles, and the kinetics and mechanisms of DNA release. We controlled the magnitude of DNA release and achieved controlled release by using mixed systems and changing the CL/PS ratio in the solution where the particles were formed. The Rose Bengal partition assay was applied for the first time to estimate the surface hydrophobicity of DNA gel particles. Both CL alone and in combination with PS promotes the formation of DNA gel particles that have an acute hydrophilic character, which may govern the posterior adsorption of plasma proteins and influence the bioavailability of the systems. The lack of hemolytic effect of these DNA gel particles suggests their potential application as long-term blood-contacting medical devices.