Kazimiera A. Wilk

Novel approach to long sustained multilayer nanocapsules: influence of surfactant head groups and polyelectrolyte layer number on the release of hydrophobic compounds

Nanoemulsion-templated long sustained polyelectrolyte (PE) nanocapsules (average size < 200 nm) loaded with Oil Red O and two cyanine-type photosensitizers: IR-786 and IR-780 were successfully fabricated by using the layer-by-layer (LbL) technique. All nanoproducts were subjected to in vitro release characteristics and analysis of selected control parameters, i.e., type of surfactant head group, characteristic release time and surfactant–polyelectrolyte interactions. Their properties were characterized by means of dynamic light scattering (DLS) compared with scanning electron microscopy (SEM) and atomic force microscopy (AFM). In our studies for construction of oil-in-water nanoemulsion templates we selected three cationic surfactants with different nature of hydrophilic head groups, i.e., double-headed (or so-called dicephalic-type) N,N-bis[3,3′(trimethylammonio)propyl]dodecanamide dimethylsulfate (C12(TAPAMS)2), bulky saccharide-derived 2-(dodecyldimethylammonio)ethylglucoheptonamide bromide (D2GHA-12) and a classic dodecyltrimethylammonium bromide (DTABr) for comparison. The polyelectrolytes were the following: polyanion of poly(sodium 4-styrenesulfonate) (PSS) and polycation of poly(diallyldimethylammonium chloride) (PDADMAC). The in vitro release profile features, studied spectrophotometrically, were interpreted in the framework of diffusion-controlled processes and stability of the first interfacial PE–surfactant complex. Accordingly, the multicharge and bulky structure of the surfactant are found to be the most desirable factors for fabrication of long sustained and stable nanocapsules encapsulating a hydrophobic active substance.

Biocompatible long-sustained release oil-core polyelectrolyte nanocarriers: From controlling physical state and stability to biological impact

It has been generally expected that the most applicable drug delivery system (DDS) should be biodegradable, biocompatible and with incidental adverse effects. Among many micellar aggregates and their mediated polymeric systems, polyelectrolyte oil-core nanocarriers have been found to successfully encapsulate hydrophobic drugs in order to target cells and avoid drug degradation and toxicity as well as to improve drug efficacy, its stability, and better intracellular penetration. This paper reviews recent developments in the formation of polyelectrolyte oil-core nanocarriers by subsequent multilayer adsorption at micellar structures, their imaging, physical state and stability, drug encapsulation and applications, in vitro release profiles and in vitro biological evaluation (cellular uptake and internalization, biocompatibility). We summarize the recent results concerning polyelectrolyte/surfactant interactions at interfaces, fundamental to understand the mechanisms of formation of stable polyelectrolyte layered structures on liquid cores. The fabrication of emulsion droplets stabilized by synergetic surfactant/polyelectrolyte complexes, properties, and potential applications of each type of polyelectrolyte oil-core nanocarriers, including stealth nanocapsules with pegylated shell, are discussed and evaluated.

New approach to hydrophobic cyanine-type photosensitizer delivery using polymeric oil-cored nanocarriers: Hemolytic activity, in vitro cytotoxicity and localization in cancer cells

We report on encapsulation of cyanine IR-768 in oil-in-water (o/w) microemulsion, i.e. fabrication of templated polymeric nanocapsules as effective nanocarriers for a new generation of photodynamic agents suitable for photodynamic therapy (PDT). Discussed here are nanocapsule imaging, their in vitro biological evaluation, cyanine encapsulation potential, and the cellular localization of cyanine IR-768 delivered in the nanocapsules to MCF-7 cancer cells. Oil-cored poly(n-butyl cyanoacrylate) (PBCA) nanocapsules were prepared by interfacial polymerization in o/w microemulsions formed by the nonionics Tween 80 (polysorbate 80, polyoxyethylene 20 sorbitan monooleate), and Brij 96 (polyoxyethylene 10 oleyl ether). Iso-propyl myristate (IPM), ethyl oleate (EOl), iso-octane (IO), and oleic acid (OA) were used as the oil phases and iso-propanol (IP) and propylene glycol (PG) as the cosurfactants. Such o/w droplets, also containing hydrophobic IR-768 in the oil phase, were applied in the interfacial polymerization of n-butyl cyanoacrylate at 10 degrees C at pH 5.0. The isolated cyanine-loaded nanoparticles were visualized by atomic force microscopy (AFM) and scanning electron microscopy (SEM), which proved their unimodal size distribution and spherical shape, with diameters dependent upon the monomer content and the template type. The entrapment efficiency of cyanine increased with increasing n-butyl cyanoacrylate concentration and varied from 65.7% to 91.7%. The results of in vitro erythrocyte hemolysis and the cell viability of breast cancer MCF-7 cells showed that the PBCA nanocapsules are quite safe carriers of IR-768 in the circulation, having a very low hemolytic potential and being non-toxic to the studied cells. Fluorescence microscopy visualized the cyanine intracellular distribution and, furthermore, demonstrated that PBCA-nanocarriers effectively delivered the IR-768 molecules to the MCF-7 doxorubicin-sensitive and -resistant cell lines. Photoirradiation of the cancer cells with entrapped photosensitizer decreased cell viability, demonstrating that this effect may be utilized in PDT.

Microstructure and structural transition in microemulsions stabilized by aldonamide-type surfactants.

Significant efforts were undertaken to characterize the microstructure and structural properties of water-in-oil (w/o), oil-in-water (o/w), and bicontinuous (bc) microemulsions composed of N-alkyl-N-methylgluconamides (n-alkyl = n-C(12)H(25), n-C(14)H(29), n-C(16)H(33)) and n-alcohols (ethanol, n-propanol, n-butanol) or iso-alcohols (iso-propanol, iso-butanol) as cosurfactants, as well as iso-octane and water. The internal structure of so created four-component system was elucidated by means of an analysis of isotropic area magnitudes in phase diagrams and conductivity measurements. Dynamic light scattering (DLS) measurements provided the microemulsion size and polydispersity. Polarity and viscosity of microemulsion microenvironment were acquired by means of electron paramagnetic resonance (EPR), UV-vis absorption spectroscopy (in the case of w/o droplets), and steady-state fluorescence (SSF) (in the case of o/w droplets). The results show that both the surfactant and the cosurfactant types affect the shape and extent of microemulsions. The size of droplets depends strongly on the type of examined microemulsion and the type of cosurfactant (linear or brunched) but is almost independent of the length of the surfactant alkyl chain. The size of microemulsion droplets ranges from 8.1 to 22.6 nm and from 3.7 to 14.3 nm respectively, for o/w and o/w microemulsions, making them good candidates for both template-based reactions and household components solubilizing media.

Nanoemulsion-templated multilayer nanocapsules for cyanine-type photosensitizer delivery to human breast carcinoma cells.

There is great clinical interest in developing novel nanocarriers for hydrophobic cyanine dyes used as photosensitizing agents in photodynamic therapy (PDT). In the present study we have employed nanoemulsion-templated oil-core multilayer nanocapsules as robust nanocarriers for a cyanine-type photosensitizer IR-786. These nanoproducts were fabricated via layer-by-layer (LbL) adsorption of oppositely charged polyelectrolytes (PEs), i.e., anionic PSS and cationic PDADMAC on nanoemulsion liquid cores created by dicephalic or bulky saccharide-derived cationic surfactants. All nanocapsules, with different thicknesses of the PE shell and average size <200 nm (measured by DLS) demonstrated good capacity for IR-786 encapsulation. The nanocarriers were visualized by SEM and AFM and their photo-induced anticancer effect and cellular internalization in human breast carcinoma MCF-7/WT cells were determined. Biological response of the cell culture, expressed as dark and photocytotoxicity as well as fluorescence of drug molecules loaded in the multilayer vehicles, analyzed by the FACS and CLSM techniques, have indicated that the delivered IR-786 did not aggregate inside the cells and could, therefore, act as an effective third-generation photosensitizing agent. In vitro biological experiments demonstrated that the properties of studied nanostructures depended upon the PE type and the envelope thickness as well as on the surfactant architecture in the nanoemulsion-based templates employed for the nanocapsule fabrication. Similarity of results obtained for stored (three weeks in the dark at room temperature) and freshly-prepared nanocapsules, attests to viability of this stable, promising drug delivery system for poorly water-soluble cyanines useful in PDT.

Assessment of extracts of Helichrysum arenarium, Crataegus monogyna, Sambucus nigra in photoprotective UVA and UVB; photostability in cosmetic emulsions

The aim of our study was to investigate the photoprotective activity and photostability efficacy of sunscreen formulations containing Helichrysum arenarium, Sambucus nigra, Crataegus monogyna extracts and their combination. UV transmission of the emulsion films was performed by using diffuse transmittance measurements coupling to an integrating sphere. In vitro photoprotection and photostability efficacy were evaluated according to the following parameters: sun protection factor (SPF), UVA protection factor (PF-UVA), UVA/UVB ratio and critical wavelength (λc) before and after UV irradiation. The results obtained show that the formulations containing polyphenols fulfill the official requirements for sunscreen products due to their broad spectrum of UV protection combined with their high photostability and remarkable antioxidant properties. Therefore H. arenarium, S. nigra, C. monogyna extracts represent useful additives for cosmetic formulation.

Biocompatible microemulsions of dicephalic aldonamide-type surfactants: Formulation, structure and temperature influence

The temperature effects upon microemulsion systems composed of dicephalic N-dodecyl-N,N-bis[(3-D-aldonylamido)propyl]amines C12-DX (gluconyl GA or lactobionyl LA)/iso-butanol/hydrophilic (diethylene glycol monoethyl ether) or hydrophobic (iso-octane) oils/water were investigated by evaluating isotropic area magnitudes in the pseudoternary phase diagrams, as well as droplet characteristics by electron paramagnetic resonance (EPR) and dynamic light scattering (DLS) spectroscopies at 25, 40 and 55 degrees C. We concluded that in the examined systems a cosurfactant, such as middle-chain alcohol, was needed to obtain large mesophase isotropic areas. The phase behavior and structure of the examined systems were temperature insensitive but they were intimately determined by the nature of the C12-DX and the polarity of the oil phase. By adjusting the nature of the oil, as well as the surfactant hydrophilicity, the performed isotropic systems containing low amounts of nonaggressive surfactant could be formulated successfully. Interfacial properties and the dynamic structure of the surfactant/cosurfactant monolayer were studied by the spin probe technique using the 16-doxylstearic acid methyl ester (16-DSE) as the appropriate probe. The polarity of the interface was not affected by temperature but the interface rigidity was dependent upon the nature of the surfactant and oil as well as on temperature. The size of the dispersed domains, evaluated by dynamic light scattering (DLS), was found to be a function of temperature, surfactant content and type of additives. The investigated o/w microemulsions (i.e., ranging from 3.0 to 8.8 nm) constituted promising templates for a variety of syntheses of nanostructures with small size and high-capacity solubilizing media.

Surface-active, micellar, and antielectrostatic properties of bis-ammonium salts.

A series of new cationic surfactants, bis-quaternary ammonium dibromides, were synthesized. The properties of these surfactants were characterized by surface tension measurements, conductivity study, and dynamic light scattering. The surface properties, such as surface excess concentration, Gamma(cmc), surface area demand per molecule, A(min), efficiency in surface tension reduction, pC(20), the effectiveness of surface tension reduction, gamma(min), critical micelle concentration, cmc, and standard free energy of micellization, DeltaG(mic)(0) and DeltaG(ads)(0), were obtained by means of surface tension measurements. The antielectrostatic properties were investigated by measuring the surface resistance, the half-charge decay time, and the maximum voltage induced on polyethylene films and propylene unwoven fabrics. Critical micellar concentrations (cmc) of investigated salts were much lower than those of the monomeric salt DTAC. The results obtained in this work were compared to analogous bis-quaternary ammonium dichlorides and well-known gemini cationic surfactants. It was found that the type of counterion had an impact on the values of the micelle ionization degree and the hydrodynamic diameters. Larger aggregates were found for bis-quaternary ammonium dibromides, which had smaller values of the ionization micelle degree than bis-quaternary ammonium dichlorides. Moreover, the antielectrostatic effect strongly depended on the kind of counterion. All investigated salts had very good antistatic properties.

Synthesis and hydrolysis of chemodegradable cationic surfactants containing the 1,3-dioxolane moiety

In acid-catalyzed reactions of long-chain aliphatic aldehydes (Ia-d wherea=n-C7H15;b=n-C9H19;c=n-C11H23; d=n-C13H27), and tridecan-7-on (Ie) with 3-chloro-1,2-propane-diol, 2-alkyl- and 2,2-dihexyl-4-chloromethyl-1,3-dioxolanes (IIa-e) were obtained. They were reacted with anhydrous dimethylamine to obtain, respectively, 2-alkyl-and [(2,2-dihexyl-1,3-dioxolan-4-yl)methyl] dimethylamines (IIIa-e), which were quaternized with methyl bromide to obtain the desired ammonium bromides (IVa-e). The structure and purity of the compounds was proved by mass spectrometry and proton nuclear magnetic resonance spectroscopy. Additionally, [(2-methyl-1,3-dioxolan-4-yl)methyl] trimethylammonium bromide and [(2,2-dimethyl-1,3-dioxolan-4-yl)-methyl] trimethylammonium bromide were synthesized as nonaggregating standards. Hydrolysis reactions of the synthesized ammonium bromides were performed by 0.1 M hydrochloric acid in 1∶1 (vol/vol) 1,4-dioxane-water mixtures at 50, 60 and 70°C. Rate constants of hydrolysis reactions were determined by observing carbonyl group formation at 280 nm. The hydrolytic reactivity of the studied surfactants (IVa-c,e) was determined under unaggregated conditions. Compound IVd showed decreased reactivity. The length of the 2-alkyl group has a minor effect on rate constant values. The influence of various substituents at the C-4 atom of the 2-nonyl-1,3-dioxolan-4-yl derivatives on rate constants was also investigated.

Polymeric micelles for enhanced Photofrin II® delivery, cytotoxicity and pro-apoptotic activity in human breast and ovarian cancer cells

BACKGROUND Searching for photodynamic therapy (PDT) - effective nanocarriers which enable a photosensitizer to be selectively delivered to tumor cells with enhanced bioavailability and diminished dark cytotoxicity is of current interest. The main objective of this study is to evaluate newly designed mixed polymeric micelles based on Pluronics P123 and F127 for the improved delivery of Photofrin II(®) (Ph II(®)) to circumvent unfavorable effects overcoming multidrug resistance (MDR) in tumor cells - in breast MCF-7/WT (caspase-3 deficient) and ovarian SKOV-3 (resistant to chemotherapy). METHODS Ph II(®)-loaded micelles were obtained and analyzed for size and morphology, solubilization efficiency, physical stability and in vitro drug release. Intracellular uptake, reactive oxygen species (ROS) generation, mitochondrial oxidoreductive potential and proapoptotic activity (TUNEL assay) studies were evaluated in the examined cancer cells. The preliminary biocompatibility characteristics of all nanocarriers was determined by assessment of their hemolytic activity in human erythrocytes and dark toxicity in cancer cells. RESULTS Dynamic light scattering (DLS) and atomic force microscopy (AFM) confirmed that almost monodisperse, sphere-shaped and nanosized (DH<20 nm) carriers were developed. Biological studies after photodynamic reaction (PDR) with encapsulated Ph II(®) revealed increased ROS level, malondialdehyde (MDA) concentration and protein damage in SKOV-3 and MCF-7/WT cells in comparison to treatment with free Ph II(®). Numerous apoptotic cells were detected after nano-therapy in both cell lines, with observed significant morphological disorders in ovarian cancer cells. In the case of encapsulated Ph II(®) only negligible disruption of human erythrocytes and cancer cells was observed. CONCLUSIONS The obtained biocompatible long-lasting nanocarriers significantly enhance the Photofrin II(®) photodynamic effect and apoptosis in both SKOV-3 and MCF-7/WT cell lines.