Ana M. Carmona-Ribeiro

Cationic Antimicrobial Polymers and Their Assemblies

Cationic compounds are promising candidates for development of antimicrobial agents. Positive charges attached to surfaces, particles, polymers, peptides or bilayers have been used as antimicrobial agents by themselves or in sophisticated formulations. The main positively charged moieties in these natural or synthetic structures are quaternary ammonium groups, resulting in quaternary ammonium compounds (QACs). The advantage of amphiphilic cationic polymers when compared to small amphiphilic molecules is their enhanced microbicidal activity. Besides, many of these polymeric structures also show low toxicity to human cells; a major requirement for biomedical applications. Determination of the specific elements in polymers, which affect their antimicrobial activity, has been previously difficult due to broad molecular weight distributions and random sequences characteristic of radical polymerization. With the advances in polymerization control, selection of well defined polymers and structures are allowing greater insight into their structure-antimicrobial activity relationship. On the other hand, antimicrobial polymers grafted or self-assembled to inert or non inert vehicles can yield hybrid antimicrobial nanostructures or films, which can act as antimicrobials by themselves or deliver bioactive molecules for a variety of applications, such as wound dressing, photodynamic antimicrobial therapy, food packing and preservation and antifouling applications.

Biomimetic nanoparticles: preparation, characterization and biomedical applications

Mimicking nature is a powerful approach for developing novel lipid-based devices for drug and vaccine delivery. In this review, biomimetic assemblies based on natural or synthetic lipids by themselves or associated to silica, latex or drug particles will be discussed. In water, self-assembly of lipid molecules into supramolecular structures is fairly well understood. However, their self-assembly on a solid surface or at an interface remains poorly understood. In certain cases, hydrophobic drug granules can be dispersed in aqueous solution via lipid adsorption surrounding the drug particles as nanocapsules. In other instances, hydrophobic drug molecules attach as monomers to borders of lipid bilayer fragments providing drug formulations that are effective in vivo at low drug-to-lipid-molar ratio. Cationic biomimetic particles offer suitable interfacial environment for adsorption, presentation and targeting of biomolecules in vivo. Thereby antigens can effectively be presented by tailored biomimetic particles for development of vaccines over a range of defined and controllable particle sizes. Biomolecular recognition between receptor and ligand can be reconstituted by means of receptor immobilization into supported lipidic bilayers allowing isolation and characterization of signal transduction steps.

Novel Formulations for Antimicrobial Peptides

Peptides in general hold much promise as a major ingredient in novel supramolecular assemblies. They may become essential in vaccine design, antimicrobial chemotherapy, cancer immunotherapy, food preservation, organs transplants, design of novel materials for dentistry, formulations against diabetes and other important strategical applications. This review discusses how novel formulations may improve the therapeutic index of antimicrobial peptides by protecting their activity and improving their bioavailability. The diversity of novel formulations using lipids, liposomes, nanoparticles, polymers, micelles, etc., within the limits of nanotechnology may also provide novel applications going beyond antimicrobial chemotherapy.

Effects of synthetic lipids on solubilization and colloid stability of hydrophobic drugs.

Aqueous miconazole (MCZ) aggregates were solubilized and/or colloidally stabilized by bilayer-forming synthetic amphiphiles such as dioctadecyldimethylammonium bromide (DODAB) or sodium dihexadecylphosphate (DHP) dispersions. Particle sizing, light absorption and scattering from drug particles, zeta-potential determination, and drug aggregation kinetics from turbidity changes in the presence or absence of lipid dispersions were obtained over a range of drug and lipid concentrations. The very low solubility of MCZ in water made possible the determination of size distributions for drug particles in water and comparison to those in the presence of DODAB or DHP nanosized bilayer fragments or entire and closed bilayer vesicles. Large drug aggregates disappeared upon incubation with nanosized bilayer fragments produced by ultrasonic dispersion with tip. Light-absorption spectra for MCZ in a poor solvent (water), in a good organic solvent (methanol), and in different lipid dispersions showed that solubilization depended on the presence of bilayer fragments. MCZ was poorly soluble in dispersions formed of closed bilayers (vesicles) of DODAB or DHP in the gel state and in phosphatidylcholine (PC) vesicles in the liquid-crystalline state. Increased hydrophobicity at the borders of bilayer fragments explained MCZ solubilization. At [MCZ]>0.4 mM, kinetics of drug aggregation, zeta-potential measurements, and size minimization were obtained upon addition of minute amounts of oppositely charged bilayer fragments ([DHP]=0.05 mM), making possible determination of a remarkable stabilizing effect of drug particles by coverage with anionic bilayer fragments. High drug colloid stability in the presence of charged bilayer fragments was achieved by two different means: (1). at large drug concentrations and small concentrations of bilayer fragments, coverage of large drug particles with bilayer fragments; (2). at large amounts of bilayer fragments, drug solubilization in its monomeric form at the borders of bilayer fragments. Inexpensive, synthetic bilayer fragments offered a large area of hydrophobic nanosurfaces dispersed and electrostatically stabilized in water, opening new prospects for drug solubilization and colloid stabilization of insoluble drug particles.

Novel immunoadjuvants based on cationic lipid: Preparation, characterization and activity in vivo.

The interactions between three different protein antigens and dioctadecyldimethylammonium bromide (DODAB) dispersed in aqueous solutions from probe sonication or adsorbed as one bilayer onto particles was comparatively investigated. The three model proteins were bovine serum albumin (BSA), purified 18 kDa/14 kDa antigens from Taenia crassiceps (18/14-Tcra) and a recombinant, heat-shock protein hsp-18 kDa from Mycobacterium leprae. Protein-DODAB complexes in water solution were characterized by dynamic light scattering for sizing and zeta-potential analysis. Cationic complexes (80-100 nm of mean hydrodynamic diameter) displayed sizes similar to those of DODAB bilayer fragments (BF) in aqueous solution and good colloid stability over a range of DODAB and protein concentrations. The amount of cationic lipid required for attaining zero of zeta-potential at a given protein amount depended on protein nature being smaller for 18 kDa/14 kDa antigens than for BSA. Mean diameters for DODAB/protein complexes increased, whereas zeta-potentials decreased with NaCl or protein concentration. In mice, weak IgG production but significant cellular immune responses were induced by the complexes in comparison to antigens alone or carried by aluminum hydroxide as shown from IgG in serum determined by ELISA, delayed type hypersensitivity reaction from footpad swelling tests and cytokines analysis. The novel cationic adjuvant/protein complexes revealed good colloid stability and potential for vaccine design at a reduced DODAB concentration.

Lipid bilayer fragments and disks in drug delivery.

Certain lipids can be dispersed as colloidally stable bilayer fragments (BF) or disks with interesting properties for solubilization and delivery of hydrophobic or amphiphilic drugs. They were first observed and characterized as such in the nineties but remained silent in the literature regarding applications for drug delivery. Only recently their potential for delivery of hydrophobic drugs started to be realized. This review deals with electrostatically or sterically stabilized bilayer fragments which provided excellent solubilization sites for antifungal drugs, acted as drugs themselves against bacteria or fungus, could be loaded with amphiphilic drugs or produced lipid-covered drug particles to be delivered as a synergistic formulation in vivo.

Salt-induced aggregation and fusion of dioctadecyldimethylammonium chloride and sodium dihexadecylphosphate vesicles

Small dioctadecyldimethylammonium chloride (DODAC) vesicles prepared by sonication fuse upon addition of NaCl as detected by several methods (electron microscopy, trapped volume determinations, temperature-dependent phase transition curves, and osmometer behavior. In contrast, small sodium dihexadecyl phosphate (DHP) vesicles mainly aggregate upon NaCl addition as shown by electron microscopy and the lack of osmometer behavior. Scatter-derived absorbance changes of small and large DODAC or DHP vesicles as a function of time after salt addition were obtained for a range of NaCl or amphiphile concentration. These changes were interpreted in accordance with a phenomenological model based upon fundamental light-scattering laws and simple geometrical considerations. Short-range hydration repulsion between DODAC (or DHP) vesicles is possibly the main energy barrier for the fusion process.

Bilayer-forming synthetic lipids: drugs or carriers?

Since their introduction as bilayer-forming synthetic compounds in the eighties, dioctadecyldimethylammonium (DODA) and dihexadecylphosphate (DHP) salts have found many uses in strategic, applied areas. In particular, DODA chloride or bromide vesicles interacted with negatively charged prokaryotic or eukaryotic cells, yielding adsorption isotherms of high affinity for the cell surface, causing cell adhesion and flocculation, changing the cell surface charge from negative to positive, and causing loss of cell viability over DODA concentration ranges that depended on the cell type being tested. This work reviews data on DODA effects on cell viability (bacteria, fungus and cultured mammalian cells) to propose DODA salts as effective anti-microbial agents that exhibit differential cytotoxicity in vitro and, therefore, deserve to be investigated as potential drugs. The full utility of these inexpensive synthetic bilayers and bilayer fragments able to act as drugs themselves and, simultaneously, as drug, gene or vaccine carriers remains hitherto unexplored.

Antimicrobial Particles from Cationic Lipid and Polyelectrolytes

Hybrid nanoparticles from cationic lipid and polymers were prepared and characterized regarding physical properties and antimicrobial activity. Carboxymethylcellulose (CMC) and polydiallyldimethylammonium chloride (PDDA) were sequentially added to cationic bilayer fragments (BF) prepared from ultrasonic dispersion in water of the synthetic and cationic lipid dioctadecyldimethylammonium bromide (DODAB). Particles thus obtained were characterized by dynamic light-scattering for determination of z-average diameter (Dz) and zeta-potential (zeta). Antimicrobial activity of the DODAB BF/CMC/PDDA particles against Pseudomonas aeruginosa or Staphylococcus aureus was determined by plating and CFU counting over a range of particle compositions. DODAB BF/CMC/PDDA particles exhibited sizes and zeta-potentials strictly dependent on DODAB, CMC, and PDDA concentrations. At 0.1 mM DODAB, 0.1 mg/mL CMC, and 0.1 mg/mL PDDA, small cationic particles with Dz = 100 nm and zeta = 30 mV were obtained. At 0.5 mM DODAB, 0.5 mg/mL CMC and 0.5 mg/mL PDDA, large cationic particles with Dz = 470 nm and zeta = 50 mV were obtained. Both particulates were highly reproducible regarding physical properties and yielded 0% of P. aeruginosa viability (10(7) CFU/mL) at 1 or 2 microg/mL PDDA dissolved in solution or in form of particles, respectively. 99% of S. aureus cells died at 10 microg/mL PDDA alone or in small or large DODAB BF/CMC/PDDA particles. The antimicrobial effect was dependent on the amount of positive charge on particles and independent of particle size. A high microbicide potency for PDDA over a range of nanomolar concentrations was disclosed. P. aeruginosa was more sensitive to all cationic assemblies than S. aureus.

Characterization of DODAB/DPPC vesicles

Dioctadecyldimethylammonium bromide (DODAB)/dipalmitoylphosphatidylcholine (DPPC) large and cationic vesicles obtained by vortexing a lipid film in aqueous solution and above the mean phase transition temperature (T(m)) are characterized by means of determination of phase behaviour, size distribution, zeta-potential analysis and colloid stability. The effect of increasing % DODAB over the 0-100% range was a nonmonotonic phase behaviour. At 50% DODAB, the mean phase transition temperature and the colloid stability were at maximum. There is an intimate relationship between stability of the bilayer structure and colloid stability. In 1, 50 and 150mM NaCl, the colloid stability for pure DPPC or pure DODAB vesicles was very low as observed by sedimentation or flocculation, respectively. In contrast, at 50% DODAB, remarkable colloid stability was achieved in 1, 50 or 150mM NaCl for the DODAB/DPPC composite vesicles. Vesicle size decreased but the zeta-potential remained constant with % DODAB, due to a decrease of counterion binding with vesicle size. This might be important for several biotechnological applications currently being attempted with cationic bilayer systems.