Some DODAB supramolecular assemblies: DODAB bilayer fragments (BF) or large closed vesicles (LV), antimicrobial peptide gramicidin D (Gr) and its assemblies with DODAB BF or DODAB LV and DODAB molecules in PMMA biocompatible polymer.
Dioctadecyldimethylammonium bromide (DODAB) is a quaternary ammonium surfactant (Quat) with interesting properties and applications. In this chapter, DODAB characteristics as compared to other Quats emphasize its self-assembly in aqueous solutions and the novel applications involving this useful cationic lipid so easily combined with biomolecules and interfaces to yield a wide range of novel uses in many fields such as delivery of drugs, vaccines and genes, design of nanoparticles, modification of interfaces, and many others yet to come.
- quaternary ammonium surfactants
- self-assembly in water
- cationic lipid in novel applications
1. The quaternary ammonium surfactants (Quats)
The quaternary ammonium surfactants or “Quats” encompass many individual chemicals [1, 2]. They are present in thousands of end-use formulations, many of which are blends of various Quats . Common uses include disinfection, detergency, fabric softening, antistatic, and wood preservation . The chemical structure determines their chemical behavior and utility. Quats will be strongly cationic due to their quaternary and positively charged nitrogen able to attach to surfaces, both organic and inorganic . With remarkable chemical stability, they can exhibit long-lasting biocidal effects . They attract anions, for example, soaps, detergents and hard water constituents, for example, carbonate and sulfate . They are attracted by negatively charged cells such as bacteria or fungus and become attached to them eventually causing their cytoplasmic membrane to leak with membrane damages leading to antimicrobial effects [6–9]. Certain Quats will biodegrade and the biodegradability decreases with increase in their alkyl chain length [10, 11]. The degradation takes place via partitioning to sludge and processing by biodegradation. The complex Quats biodegradation occurs in several steps and depends on the Quat chemical structure, Quat interactions with the sludge determining adsorption and desorption, microorganisms present in the sludge and the presence or absence of anions; alkylammonium surfactants chemically modified with biological moieties such as carbohydrates, amide, aminoacids or peptides were better degraded . From the point of view of Quats synthesis, compounds bearing more than one positive charge were readily obtained at economical cost from compounds with at least two tertiary amines that could be readily quaternized; some of these displayed potent antibacterial and antibiofilm activity and did not trigger bacterial resistance systems as those from methicillin-resistant Staphylococcus aureus (MRSA); mono-Quats and several bis-, tris- and tetra-Quats tested against bacteria within a few hundred generations yielded a lack of resistance for Quats of higher charge when compared to mono-Quats .
Quats chemical structure determines their self-assembly in water solution. The theory for the self-assembly of dilute surfactant solutions is well established and very successful [14, 15]. This theory applies also to Quats since their amphiphilic molecular nature includes polar and apolar regions in the same molecule. The theory relates the self-assembly in water solution with the geometric parameter v/al. The definition of v/al is given by v, the volume of the hydrocarbon region of the surfactant; a, the optimal head group area, and l, as the optimal hydrocarbon chain length related to its maximum extended length. One should notice that the nature and shape of the assemblies are intimately related to the v/al value. For instance, in the case of spherical micelles, v/al < 1/3 whereas for vesicles or bilayers, ½ < v/al ≤ 1. When bilayer vesicles are the desired structure, larger v is required as is the case of the double-chained surfactants. Single-chained surfactants and lower v are required for micellar structures. For example, a single-chained quaternary ammonium surfactant such as cetyltrimethylammonium bromide (CTAB) has a lower v than the corresponding double-chained quaternary ammonium surfactant. The self-assembly of CTAB and dioctadecyldimethylammonium bromide [DODAB] from calculations for their respective geometric parameters predicts, as indeed observed, CTAB molecules assembling as micelles and DODAB molecules assembling as bilayers in water solutions.
Not only the molecular geometry of the Quats determines their assembly in water solution: specific counterion effects also do . Counterion adsorption and Stern layer effects change the optimal headgroup area a. In general, counterions will adsorb to some extent to the surfactant headgroups. Specific interactions of a nonelectrostatic origin like dehydration or hydration of the surfactants, conformational changes in the surfactant headgroup, size of the adsorbed counterion are important because they determine the thickness of the Stern layer and the actual surface potential. Specific counterions can change the lateral interactions between surfactants in a micelle, monolayer or bilayer. By means of the direct force measurement technique developed by Israelachvili  after depositing DODAB bilayers with the Langmuir-Blodgett technique on two molecularly smooth mica surfaces and bringing these surfaces together in an aqueous solution, the measurements of the interaction forces between the bilayers as a function of their separation a repulsive double-layer force are experienced. Fitting the measured double-layer force with theory allows the surface potential to be estimated, from which the binding affinity of the ions can be determined . Apart from the repulsive double-layer interaction, the van der Waals interaction and possibly the ion-ion correlation interaction, which are both attractive, must be taken into account . The interactions between bilayers of dihexadecyldimethylammonium acetate and bromide surfactants, which are soluble in water and adsorbed from solution as a bilayer onto the mica surfaces, were determined by Pashley and coworkers . Marra employed the Langmuir-Blodgett deposition technique for an insoluble surfactant like DODAB so that the solution did not contain any aggregates and the binding of anions to the quaternary ammonium headgroups would not depend sensitively on the precise length of the hydrocarbon tails . The anions investigated bound to the headgroups following a lyotropic series where the least hydrated, smallest anions bound with highest affinity . Lateral interactions between DODAB adjacent molecules in a monolayer at the air-water interface and interactions between bilayers of DODAB surfactants exhibited a pronounced ion specificity. Large hydrated counterions like the fluoride, hydroxide, and acetate ions gave expanded monolayer compression isotherms. Fluoride, hydroxide, and acetate counterions did not bind to DODAB headgroups. Following the lyotropic series for anion sizes F-> C1-> Br-, the smaller the (hydrated) anion, the more contracted the monolayer . For dioctadecyldimethylammonium (DODA) acetate, chloride or bromide, vesicle size and zeta-potentials were inversely related; an increase in the zeta-potential was accompanied by a decrease in vesicle size, in accordance with the self-assembly theory; DODA acetate bilayer vesicles had the largest, less tightly bound and more hydrated counterion and exhibited the smallest size in comparison with those obtained from the other DODA salts .
2. DODAB hybrid assemblies
DODAB remarkable interactive capability with opposite charges of silica particles [20–23], silicon wafers , polymeric particles [25–31]; polymer films [32–34], drugs [35–45], nucleic acids [31, 46], oligonucleotides [47–49], proteins [30, 50–54], peptides [9, 55–57], polyelectrolytes [8, 9, 36, 58, 59] and many other important surfaces, biological cells, molecules and nanostructures [60–67] is at the root of DODAB popularity in the literature spanning a huge variety of subjects. Today (December 10th, 2016) a search in American Chemical Society, PubMed and Scopus databases retrieved 104, 140 and 1208 documents, respectively, quoting DODAB. Therefore, this review just gives an overview on DODAB recent possibilities, and many others have already appeared or are yet to come.
The interaction between DODAB and solid surfaces like silicon wafers depends on the charge density of the solid surface, which depends on the nature and concentration of bound counterions and DODAB ability to displace them; the cation more tightly bound to the negatively charged surface solid surface should be Li+ that would be difficult to displace by the DODAB cation, in contrast to the loosely bound Cs+ with its large ion radius and low charge density. In summary, DODAB adsorption proceeded in accordance with charge density on the solid surface thus depending on nature and concentration of counterions and DODAB ability to displace them; increasing the ionic strength increases silanol dissociation, surface charge density, and DODAB adsorption . The effect of monovalent salt nature and concentration over a range of low ionic strengths (0–10 mM LiCl, NaCl, KCl, or CsCl) and at two different pH values (6.3 and 10.0) on DODAB adsorption onto flat SiO2 surfaces evaluated by in situ ellipsometry. This technique allowed precise evaluation of thin film thicknesses on very smooth solid surfaces such as those of silicon wafers. Thereby, DODAB adsorption isotherms of high affinity showed adsorption maxima consistent with bilayer deposition only around 10 mM monovalent salt at both pH values. In contrast, when pure water was the intervening medium, DODAB adsorption decreased substantially. The nature of counterion on the charged solid surface was also important to determine DODAB adsorption: at 10 mM CsCl or LiCl, the highest and the lowest affinity constants for DODAB adsorption onto SiO2 were, respectively, obtained . This was understandable from the fact that DODAB adsorption onto the solid surface required as a first step the displacement and cation exchange at the solid surface. DODAB adsorption consistently followed the expected facility of cation exchange at the surface required for DODAB adsorption. Figure 1 illustrates the effect of counterion nature and concentration on DODAB adsorption from bilayer fragments (BF) onto silicon wafers as determined from in situ ellipsometry measurements .
The changes of the electrostatic repulsion between adjacent DODAB molecules in a bilayer as the one due to interaction with counterions or oppositely charged inorganic or organic species can drastically change the nature of DODAB assemblies. For example, monovalent salt at a moderate concentration was reported to induce fusion of DODAB bilayer fragments [68–71] with induction of hydrophobic defects at the bilayer-water interface . When the electrostatic repulsion is high as in pure water or in the presence of low concentrations of poorly bound counterions, interdigitation represents a way of relaxing the intermolecular repulsion in the bilayer; adhesion between DODAB bilayers due to interdigitation between DODAB molecules in the bilayer , molecular dynamic simulations , differential scanning calorimetry (DSC), and X-ray scattering in the subgel state  further supported DODAB tendency to display hydrophobic moieties in its assemblies for relaxation of the electrostatic repulsion.
Other interesting instances refer to the formation of catanionic bilayers from DODAB and anionic oleosiloxanes  or oleic acid ; DODAB membrane fragments and fatty-acid esters of cyclosiloxanes formed dense multibilayered vesicles; the transformation took place once the ester groups hydrolyzed to yield carboxyl groups yielding the anionic silicone surfactant in situ and the catanionic system with DODAB. The oleo-silica compound was obtained via hydrosilylation of methyl undec-10-enoate with 1,3,5,7-tetra-methylcyclotetrasiloxane (1). Flat DODAB/oleic acid bilayer sheets were obtained at about 1:1 molar ratios for DODAB/oleic acid binary dispersions; the relaxation of the electrostatic repulsion between DODAB molecules in the bilayer due to the incorporation of OA into DODAB bilayer decreased the membrane curvature and increased the aggregate size; introduction of the fatty acid around equimolar ratios led to flat DODAB/OA bilayer assemblies in the dispersions . The electrostatic attraction between DODAB and anionic amphiphiles decreased the mean area per molecule, increased the geometric parameter v/al, and increased the aggregate size similarly to the fusogenic effects reported upon increasing counterion concentration [68–72, 75, 76].
Figure 2 shows cryo-transmission electron micrographs (cryo-TEM) of vitrified DODAB bilayer fragments obtained by sonication of DODAB in water , unilamellar vesicles of about 200–400 nm obtained by vaporization of a DODAB chloroform solution in water at 70 degrees centigrades (above the gel to liquid-crystalline phase transition temperature of the DODAB bilayer and above the chloroform boiling point)  and very large unilamellar DODAB vesicles from salt-induced fusion of DODAB bilayer fragments [68, 69].
Combinations of DODAB and dihexadecylphosphate (DHP) yielded miscible catanionic bilayers over a range of molar ratios, though DODAB and DHP miscibility in the bilayer domain was non-ideal . For vesicles with DODAB as the predominant lipid, small sizes, high positive zeta potential, low main transition temperature, less angular structure, good stability, and high internal water compartment contrasted with similar properties determined for the DHP-rich vesicles; DODAB improved the bilayer fluidity of DHP vesicles both in the liquid-crystalline and in the rippled bilayer phases . Interestingly, the reduction of positive charges on the DODAB/DHP vesicles improved also the survival of mammalian cells in culture . These results might become important for future drug/gene delivery applications.
Cholesterol has been suggested to play a role in stable vesicle formation by adjusting the molecular packing of the vesicular bilayer. The Langmuir monolayer approach with infrared reflection-absorption spectroscopy (IRRAS) elucidated the effects of cholesterol on molecular packing of double-chained cationic surfactants . Combining cholesterol with DXDAB monolayers at the air-water interface (X meaning the hydrocarbon chain length) reduced desorption of DXDAB with short alkyl chains, for example, ditetradecyldimethylammonium bromide or dihexadecyldimethylammonium bromide, into the water sub-phase and condensed the DXDAB monolayers . For the DODAB monolayers, cholesterol had a dual effect inducing both order and disorder of the neighboring hydrocarbon chains; the flexible alkyl side-chain of cholesterol along with the corresponding portion of neighboring hydrocarbon chains formed a fluidic region, counteracting the conformational order induced by the sterol ring of cholesterol interacting with the alkyl chains .
The effect of varying the molar proportion of DODAB and neutral dipalmitoylphosphatidylcholine (DPPC) in DODAB/DPPC vesicles revealed a high bilayer and coloidal stability with good miscibility for the binary system and absence of phase separation at a molar proportion equal to 1 . The demixing and crystallization of DODAB/DPPC binary lipid system were recently found to take place when DODAB or DPPC was dominant in the mixture (DPPC/DODAB = 1/2 or DPPC/DODAB = 2/1); when DODAB was no more than equimolar (e.g., DPPC/DODAB = 2/1 and 1/1), there was good miscibility in absence of DODAB crystallization . At high or low DODAB, DPPC molar proportions, phase separation occurred upon cooling so that gel domains rich in DODAB phase-separated from DPPC-DODAB domains or DPPC domains. This phase separation for the gels would mean demixing and crystallization originating DODAB-rich and DPPC-rich tilted gel separated domains upon incubation at low temperatures .
3. Novel applications for DODAB hybrid assemblies
Aqueous solubilization of water-insoluble materials is highly important for pharmaceuticals, detergency, emulsion polymerization, enhanced oil recovery, and textile dyeing. Among colloidal Self-assembled structures, micelles/vesicles are efficient solubilizers but the solubilization properties of bilayers of vesicles are superior [83, 84]. A series of double-chained surfactants, with increasing chain length (C12–18) mixed with single chained dodecylethyldimethylammonium bromide (DODABB) solubilized curcumin thanks to hydrophobic-hydrophobic and electrostatic interactions with preservation of curcumin antioxidant activity in food .
Aiming at the production of nanoparticles (NPs) for drug delivery, DODAB has been very useful to harmonize oppositely charged polysaccharides such as carboxymethylcellulose  or hyaluronic acid  with hydrophobic drugs such as amphotericin B , indomethacin , and tocoferol (vitamin E) . Carboxymethylcellulose/DODAB/indomethacin NPs were prepared by direct injection of DODAB/indomethacin ethanol solution into a carboxymethylcellulose water solution . Similarly, hyaluronate/soybean lecithin/DODAB/vitamin E NPs were prepared by direct injection of vitamin E/soybean lecithin/DODAB ethanol solution into hyaluronic acid water solution; further incorporation of these NPs in polymeric, bioadhesive films containing Aloe vera extract, hyaluronic acid, sodium alginate, polyethyleneoxide (PEO) and polyvinylalcohol (PVA) represented an innovative treatment for skin wounds .
A three-dimensional layer-by-layer (LbL) structure composed by xanthan and galactomannan biopolymers on DODAB liposome template created a LbL structure up to eight layers, evaluated using quartz crystal microbalance (QCM) and zeta potential analysis; these bilayer-coated NPs increased up to five times the sustained release of epidermal growth factor (EGF) and could be useful for improving the release profile of low-stability drugs like EGF .
The approach of combining important biomolecules such as proteins or nucleic acids with DODAB and further stabilizing the hybrids with hydrophilic polymers has been very useful for several biomedical and biotechnological applications. For instance, the delivery of DNA plasmids or small interference RNA (siRNA) to cells requires nanocarrier stability after in vivo administration though too strong stabilization can decrease the carrier efficiency; after characterizing DODAB/monoolein/pDNA or siRNA lipoplexes [88, 89], the nanocarriers were pegylated and tested for stability in serum and gene silencing in cultured cancer cells with promising results: pegylation avoided siRNA dissociation from the nanocarriers in human serum and improved transfection efficiency . Stable lipoplexes of small size (100–160 nm) with a positive surface charge (>+45 mV) were readily internalized by human non-small cell lung carcinoma (H1299) cells and were efficient in promoting gene silencing. Monolein had a similar gene silencing ability as the commonly used helper lipid 1,2-dioleyl-3-phosphatidylethanolamine (DOPE), but with much lower cytotoxicity . More recently, the same DODAB/monolein system was used to incorporate cell wall surface proteins (CWSP) from Candida albicans aiming at the production of an antigen delivery system (ADS) for a potential vaccine against candidiasis; the system facilitated antigen uptake by dendritic cells in vitro and induced higher levels of pro-inflammatory cytokines and opsonizing specific IgG antibodies in serum of mice immunized subcutaneously .
DODAB was also used to treat spores of Bacillus subtilis aiming at gene gun delivery of DNA plasmids in mice; DODAB treated spores allowed efficient plasmid adsorption and could be loaded into biolistic cartridges and efficiently delivered into mice for induction of specific cellular and antibody responses required for DNA vaccines in vivo .
For textile materials, sometimes modification of the wettability of hydrophobic surfaces is essential. For instance, DODAB adsorption to hydrophobic polypropylene (PP) thin films dramatically enhanced surface adsorption of different proteins from soybeans and represented a facile treatment to obtain PP-modified surfaces that were completely hydrophilic .
DODAB combinations with graphene enhanced adsorption of hydrophobic analytes and improved the design of novel sensors for phenolic compounds; graphene/DODAB films exhibited remarkable synergistic effects toward the oxidation of tetrabromobisphenol TBBPA, due to the greatly increased TBBPA accumulation in the film and magnitude of the peak currents detected by chronocoulometry . In another interesting instance, immobilization of urease for urea biosensing was achieved employing a DODAB monolayer at the air-water interface and natural exopolysaccharides from microalgae in the aqueous subphase; both DODAB and polysaccharide provided an appropriate microenvironment for the enzyme, enhanced its adsorption in the monolayer and could be used for the production of films supported on solid substrates .
Interestingly, the anisotropic polymerization of DNA adsorbed to a DODAB monolayer at the air-water interface yielded a one-dimensionally assembled belt-shaped structure and a unimolecular thickness for the polymerized DNA; thereby, the polymerization could be regulated in the two-dimensionally confined medium of the Langmuir-Blodgett film .
In another instance, DODAB monolayers allowed to ascertain the nanostructure of assembled oligonucleotides; two oligonucleotides, a 19-mer bearing thrombin binding aptamer sequence and a 21-mer with human telomeric sequence were end-labeled with fluorescent groups and their fluorescence spectra and G-quadruplex folding at DODAB monolayer interface were reported for the first time. Thanks to film balance measurements (pressure-area isotherms), the fluorescence spectra recording using a fiber optic accessory interfaced with a spectrofluorimeter and the DODAB monolayer, the fluorescence energy transfer efficiency of monolayer adsorbed probes increased significantly in the presence of sodium or potassium ion in subphase, which indicated that the probes retained their cation binding properties when adsorbed at the DODAB monolayer interface .
In the fields of antimicrobials and adjuvants for vaccines, DODAB has also been playing important roles. Biocompatible NPs of poly (methylmethacrylate) (PMMA) were synthesized in the presence of DODAB and characterized by dynamic light scattering for sizing, polydispersity and zeta potential analysis, scanning electron microscopy (SEM) for morphology visualization, and plating plus colony-forming unities (CFU) counting for the determination of antimicrobial activity; there was a high permanent load of DODAB in the NPs, and a remarkable antimicrobial activity of PMMA/DODAB NPs, which was much higher than the one determined for DODAB itself . PMMA particles loaded with DODAB were thus obtained from particle synthesis by emulsion polymerization in the presence of DODAB, a facile, fast, low-cost approach to obtaining highly efficient antimicrobial nanoparticles with a permanent DODAB load. Other hybrid DODAB assemblies with the antimicrobial peptide gramicidin (Gr) reunited the complementary antimicrobial properties of DODAB with those of the peptide . DODAB dispersed as large closed bilayer vesicles (LV) or bilayer disks (BF) was added of gramicidin (Gr), which is an antimicrobial peptide assembling as channels in membranes, increasing their permeability toward cations and displaying high toxicity against mammalian cells; DODAB/Gr bilayers exhibited microbicidal action and reduced cytotoxicity against eukaryotic cells . The novel formulations were characterized by dynamic light scattering for sizes an zeta-potentials, leakage from large vesicles induced by transmembrane gramicidin pores with dissipation of osmotic gradients, determination of lytic effects on bacteria and plating plus viable bacteria counting over a range of DODAB and/or Gr concentrations . Gr dimers reconstituted functional channels in LV and the insertion of these channels in DODAB bilayer increased the charge density for LV but did not affect charge density of BF, with Gr at the BF borders. DODAB/Gr combinations diminished the high peptide toxicity against Saccharomyces cerevisae and had the advantage of broadening the spectrum of antimicrobial activity for the combination by inducing Escherichia coli and Staphylococcus aureus lysis and bacterial death. Thereby, the cytotoxicity of the peptide against eukariotic cells was reduced, and the spectrum of antimicrobial activity was broadened since DODAB and Gr displayed complementary activities . More recently, the PMMA/DODAB and DODAB/Gr antimicrobial systems revealed potential uses in food microbiology for killing important food-borne pathogens such as Escherichia coli, Salmonella enterica, Staphylococcus aureus and Listeria monocytogenes .Nowadays, a large family of bacterial genes (generally termed quaternary ammonium genes) encode efflux pumps capable of expelling many Quat structures from bacterial cells, leading to a decrease in susceptibility to Quats . Since bacteria will inevitably find ways of resisting the existing antibiotics and Quats, maybe hybrid assemblies of antimicrobials will prove strategical to overcome resistance. Table 1 shows some schematic representations of DODAB combinations with gramicidin  or biocompatible PMMA polymer in PMMA/DODAB nanoparticles . Their antimicrobial effects against food-borne bacteria were summarized on Table 2 .
|Assembly||MBC in mM; mg/mL/reduction in log(CFU/mL)|
|E. coli||S. enterica||S. aureus||L. monocytogenes|
|Gr||0.010; 0.019/0.3||0.010; 0.019/0.5||0.010; 0.019/2.1||0.005; 0.009/7.6|
|DODAB BF||0.063; 0.039/7.6||0.500; 0.316/1.3||0.063; 0.039/3.4||0.125; 0.079/7.8|
|DODAB BF/Gr||0.031; 0.019/7.5||0.250; 0.158/0.9||0.015; 0.010/3.8||0.125; 0.079/8.0|
|DODAB LV||0.015; 0.010/4.5||0.500; 0.316/0.7||0.015; 0.010/2.9||0.250; 0.158/5.7|
|DODAB LV/Gr||0.015; 0.010/4.6||0.500; 0.316/0.4||0.031; 0.019/2.7||0.063; 0.039/6.0|
|PMMA/DODAB NPs||–; 2.500/2.2||–; 1.250/0.1||–; 5.000/3.1||–; 5.000/1.5|
In vaccine development, adjuvants and immunostimulants have the important task of presenting antigens to the immune system eliciting an amplified and antigen-specific immune response. Among the adjuvants, DODAB is especially important due to its biomimetic hybrid nanostructures with an outer DODAB coating or an inner DODAB core, which join the advantages of particles and lipids and permit a robust control over size-dependent immune responses in vivo. Recently, hybrid nanomaterials based on DODAB with potential for combination with antigens and immunostimulants for vaccine development were reviewed . For instance, in compositions with derivatives of the myco-bacterial cell wall component, the cord factor trehalose dimycolate (TDM), which is the most abundant glycolipid in the mycobacterial cell wall, DODAB yielded highly efficacious immunoadjuvant formulations for tuberculosis vaccines able to induce cell-mediated immunoresponses against intracelular bacteria [101, 102]. In general, DODAB has been combined not only with antigens of interest but also with important immunostimulants such as oligonucleotides, glycolipids or lipopeptides .
DODAB-covered particles and DODAB bilayer fragments were often used as immunoadjuvants since DODAB can both adsorb onto several hydrophobic or hydrophilic particles and present antigens (Ag) to elicit amplified immunoresponses . The electrostatic attraction drives the adsorption of a cationic DODAB bilayer onto oppositely charged polystyrene sulfate (PSS) nanoparticles (NPs) over a range of particle sizes [25, 27]. Adsorption isotherms and electrokinetic properties of the covered particles show the deposition of DODAB onto silica or PSS particles at maximal adsorption [21, 22, 25, 27, 28]. At maximal adsorption, the area per DODAB molecule adsorbed onto PSS particles is 0.286 nm2, which is half of the usual area per monomer in DODAB monolayers at the air-water interface and suggests bilayer deposition onto the polystyrene surface; electrokinetic properties of the covered particles are very similar to those of DODAB vesicles . The hydrodynamic diameter of particles in the particles/DODAB mixtures increases 9–10 nm. A tiny concentration of 10-micromolar is required for bilayer coverage of 109 particles (300 nm diameter) per mL at sub-toxic DODAB concentrations. DODAB toxicity against fibroblasts in cell culture becomes significant above 0.1 mM DODAB; there is 50% of cell death at 0.5 mM DODAB . Lipid-covered NPs are useful for antigen presentation .
The mean molecular area of DODAB in a monolayer at the air-water interface is 0.6 nm2 . For particles with 300 nm of mean diameter, the bilayer coverage of total surface area on 5 × 109 particles/mL requires 10 μM DODAB only . At this minute amount, the usual DODAB toxicity is not relevant. In contrast, DODAB vesicles used as immunoadjuvants over the millimolar range of DODAB concentrations may be toxic in vivo . Antigen (Ag) adsorption to the PSS/DODAB assembly does not disturb the order of the particulate over a range of Ag concentrations; the PSS/DODAB system at 5 × 109 particles/mL accommodates well up to 25 μg/mL Ag with narrow size distributions for PSS/DODAB/Ag NPs over this range of Ag concentrations . This homogeneity for the particle size in the dispersions yields low polydispersities determined by dynamic light scattering, inside the 0.05–0.10 range .
DODAB molecules ultrasonically dispersed in aqueous solution are nano-sized bilayer disks or bilayer fragments (BF); the electrostatic repulsion at low ionic strength keeps the BF stable in aqueous dispersions [39, 64]. DODAB BFs are antimicrobial agents [39, 43], carriers for hydrophobic drugs  and useful for the production of lipid-covered particles such as bilayer-coated sílica  or PSS . DODAB BFs also present antigens to the immune system inducing cellular immune responses . These open bilayers differ from their mother vesicles by especial features. They do not respond to osmotic gradients because they do not have an inner aqueous compartment. They have a discoidal shape with disks exhibiting one bilayer thickness and both faces available to display antigens . They have domains of fluid and gel lipid phases . They solubilize hydrophobic molecules sometimes in contrast to their mother vesicles that do not do so as in the case of amphotericin B . DODAB BF interact with proteins, oligonucleotides or DNA via both the hydrophobic effect and the electrostatic attraction at low ionic strength. Bovine serum albumin (BSA) purified 18/14 kDa antigens from Taenia crassiceps cysticerci (18/14-Tcra) or a recombinant heat-shock protein (hsp-18 kDa) from Mycobacterium leprae adsorb on DODAB BF . DODAB BF/Ag NPs are stable over a range of DODAB and Ag concentrations; these ranges vary with the Ag nature and are different for different antigens . The production of cytokines by lymph nodes (LN) cells of immunized mice in culture is important to determine the nature of immune response induced by PSS/DODAB/Ag or DODAB BF/Ag. The mice immunized with antigen alone, adjuvant/antigen or adjuvant alone provide LN cells in culture that produce different cytokines depending on Ag and adjuvant nature . A sandwich kit enzyme-linked immunosorbent assay (ELISA) determines the analytical concentrations of the cytokines produced after reestimulating the cells in culture. The cytokines profile is rather different from immunization with the parasite and the bacterium antigens . The high levels of IL-12 and IFN-gamma induced by PSS/DODAB/Ag and DODAB BF/Ag when Ag is hsp-18kDa shows that these adjuvants are useful for the design of subunit vaccines against intracelular bacteria. IL-12 and IFN-gamma are the most important cytokines in innate responses to intracellular bacteria such as M. leprae or tuberculosis; when Ag is 18/14-Tcra, there is an enhancement in production of IL-10 and Il-13 by LN cells elicited by DODAB BF/Ag. These cytokines are typically associated with responses to allergens and parasites such as helminths and mediate differentiation of CD4+-T cells into Th2 cells . On the other hand, the Mycobacterium leprae antigen carried by DODAB BF or PSS/DODAB adjuvants elicits low levels of these cytokines. Responses are indeed different for the helminthes and the bacteria antigens and antigen-specific as they should be [54, 106].
IL-10 exerts an inhibitory effect on macrophages and dendritic cells by decreasing the production of IL-12 and the expression of class II major histocompatibility complex (MHC) . Macrophages and DCs also secrete IL-12 that induces T cells differentiation into Th1 and natural killer (NK) cells with increased IFN-gamma synthesis and cytotoxic activity. The adaptive immunity against intracellular bacteria is principally cell mediated and consists of activation of macrophages by CD4+T cells as well as killing of infected cells by CD8+ cytotoxic T lymphocytes (CTL). Naïve CD4+ T cells may differentiate into distinct subsets, such as Th1 and Th2 cells in response to different antigens.
Due to its chemical stability and low cost when compared to other natural or synthetic lipids, DODAB has been intensively investigated aiming at subunit vaccine design. Major problems of liposomal formulations based on DODAB are the high DODAB concentration (1–10 mM DODAB) and the large liposomes size [52, 106, 107]. Minimization of DODAB dose is required for administration in vivo. DODAB BF effectively present antigens at 0.1 mM DODAB only; supported DODAB bilayers on PSS or silica require even lower DODAB concentrations [22, 25, 30, 54]. The total surface area on the BF dispersion available for antigen association are much larger than the one for closed, large and sometimes multibilayered liposomes. Thus, the first advantage of DODAB BF, PSS/DODAB or silica/DODAB as adjuvants would be the low DODAB concentration required for Ag presentation. The second advantage of BF is the nanosize. Depending on sonication power and time plus composition of the dispersing medium that determine colloidal stability, DODAB BF/Ag complexes have a few tenths of nanometers in size (40–80 nm). This size is effective for antigen delivery to antigen-presenting cells (APCs), generating potent and combined humoral and CD8+ T cell immunity [109–111]. Over a range of low DODAB and antigen concentrations ([DODAB] ≤0.1 mM; 0.001–0.05 mg/mL antigen), adjuvant/antigen combinations were cationic, stable, homodisperse and immunogenic at low DODAB dose, low cost, low sizes for improved dendritic cells uptake, high chemical stability, prone to present several different antigens and displaying low or even absent cytotoxicity. They were remarkably superior to alum due to their ability to elicit the cellular Th1 immune response. Contrary to alum or DODAB LV (1–10 mM DODAB), local or systemic adverse effects in mice were completely absent over the 0.1–0.01 mM DODAB range. Silica/DODAB, PSS/DODAB, and DODAB BF are available over the sub-200 nm range of sizes thus presenting potential also for design of mucosal vaccines. The third advantage of BF is the absence of depots at the site of injection, an inflammatory reaction that is not always desirable . These depots occur for DODAB large vesicles (LV) and appear due to inflammatory responses at the site of injection [107, 108]. Similar sizes for adjuvant and adjuvant-antigen complexes evidenced that the antigens readily adsorbed and stabilized the adjuvant; conversely, the adjuvant also stabilized the antigens preventing antigen-antigen aggregation as often observed for protein-protein interactions [30, 54].
An important component of the early innate immune response to viruses and bacteria is IL-12 that enhances the IFN-gamma production and the development of Th1 cells; IL-12 is involved in the combat of infections by cell-mediated immunity, for example, leishmaniasis . Subunit vaccines against protozoa that survive within macrophages require as principal defense mechanism the cell-mediated immunity, particularly directed to macrophage activation by Th1 cell-derived cytokines. Immune responses to leishmaniasis against the parasite Leishmania donovani involve cell-mediated immune response of the Th1 type and CD4+ Th1 cells activation for killing phagocytosed parasites. Leishmania-specific Th1 CD4+ T cells produce IFN-gamma, that activates macrophages to kill intracellular parasites. On the other hand, the parasite activates Th2 cells increasing their production of Th2 cytokines that suppress the activity of macrophages and increase parasite survival . Similarly, during the liver stages of malaria, CD8+ T cells kill infected hepatocytes and induce the secretion of IFN-gamma activating the production of nitric oxide and other agents by the hepatocytes for killing the parasites. IL-12 stimulates IFN-gamma production inducing resistance to sporozoite challenge in rodents and non-human primates . Il-12 also increases the cytotoxic activity of natural killer (NK) cells after viral infections thereby mediating the NK cell killing of virus-infected cells for combating the infection. Recombinant DNA vaccines expressing membrane and envelope of viral proteins may benefit from the DODAB BF or PSS/DODAB adjuvants, which can also carry DNA  or oligonucleotides .
DNA sequences containing unmethylated CpG dinucleotide generate danger signals that are recognized by the immune system; they are typical of bacteria and viruses but rare in vertebrates activating cells that express Toll-like receptor 9 and induce an innate immune response characterized by the production of Th1 cytokines . Both CpG and DODAB improve Th1 responses against antigens when used separately. DODAB BF/CpG presenting ovalbumin (OVA) also enhanced Th1 immune responses . DODAB BF/CpG/OVA also did not result in any observable depot effect at the site of prime suggesting their direct action on the antigen presenting cells (APC) of the draining LN. Only NPs can specifically target LN-resident cells . The interstitial flow convects sub-100 nm NPs into the draining lymphatic vessels; NPs are not trapped in the tissue interstitium. Nano-sizes allow direct LN targeting without the use of specific ligands. In the LN, antigen-presenting cells (APCs) rapidly capture the NPs. A few reviews are available on DODAB applications for the development of novel hybrid assemblies useful as immunoadjuvants, gene or RNA carriers [114–118].
The author thanks Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for financial support (CNPq302352/2014-7).