Correlation of the Physicochemical and Structural Properties of pDNA/Cationic Liposome Complexes with Their in Vitro Transfection

In this study, we characterized the conventional physicochemical properties of the complexes formed by plasmid DNA (pDNA) and cationic liposomes (CL) composed of egg phosphatidylcholine (EPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) (50/25/25% molar ratio). We found that these properties are nearly unaffected at the studied ranges when the molar charge ratio (R-+/-) between the positive charge from the CL and negative charge from pDNA is not close to the isoneutrality region (R-+/- = 1). However, the results from in vitro transfection of HeLa cells showed important differences when R-+/- is varied, indicating that the relationships between the physicochemical and biological characteristics were not completely elucidated. To obtain information regarding possible liposome structural modifications, small-angle X-ray scattering (SAXS) experiments were performed as a function of R-+/- to obtain correlations between structural, physicochemical, and transfection properties. The SAXS results revealed that pDNA/CL complexes can be described as being composed of single bilayers, double bilayers, and multiple bilayers, depending on the R-+/- value. Interestingly, for R-+/- = 9, 6, and 3, the system is composed of single and double bilayers, and the fraction of the latter increases with the amount of DNA (or a decreasing R-+/-) in the system. This information is used to explain the transfection differences observed at an R-+/- = 9 as compared to R-+/- = 3 and 6. Close to the isoneutrality region (R-+/- = 1.8), there was an excess of pDNA, which induced the formation of a fraction of aggregates with multiple bilayers. These aggregates likely provide additional resistance against the release of pDNA during the transfection phenomenon, reflected as a decrease in the transfection level. The obtained results permitted proper correlation of the physicochemical and structural properties of pDNA/CL complexes with the in vitro transfection of HeLa cells by these complexes, contributing to a better understanding of the gene delivery process.

Structural effect of cationic amphiphiles in diacetylenic photopolymerizable membranes

Liposomes have been an excellent option as drug delivery systems, since they are able of incorporating lipophobic and/or lipophilic drugs, reduce drug side effects, increase drug targeting, and control delivery. Also, in the last years, their use reached the field of gene therapy, as non-viral vectors for DNA delivery. As a strategy to increase system stability, the use of polymerizable phospholipids has been proposed in liposomal formulations. In this work, through differential scanning calorimetry (DSC) and electron spin resonance (ESR) of spin labels incorporated into the bilayers, we structurally characterize liposomes formed by a mixture of the polymerizable lipid diacetylenic phosphatidylcholine 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (DC8,9PC) and the zwitterionic lipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), in a 1:1 molar ratio. It is shown here that the polymerization efficiency of the mixture (c.a. 60%) is much higher than that of pure DC8,9PC bilayers (c.a. 20%). Cationic amphiphiles (CA) were added, in a final molar ratio of 1:1:0.2 (DC8,9PC:DMPC:CA), to make the liposomes possible carriers for genetic material, due to their electrostatic interaction with negatively charged DNA. Three amphiphiles were tested, 1,2-dioleoyl-3-trimetylammonium-propane (DOTAP), stearylamine (SA) and trimetyl (2-miristoyloxietyl) ammonium chloride (MCL), and the systems were studied before and after UV irradiation. Interestingly, the presence of the cationic amphiphiles increased liposomes polymerization. MCL displaying the strongest effect. Considering the different structural effects the three cationic amphiphiles cause in DC8,9PC bilayers, there seem to be a correlation between the degree of DC8,9PC polymerization and the packing of the membrane at the temperature it is irradiated (gel phase). Moreover, at higher temperatures, in the bilayer fluid phase, more polymerized membranes are significantly more rigid. Considering that the structure and stability of liposomes at different temperatures can be crucial for DNA binding and delivery, we expect the study presented here contributes to the production of new carrier systems with potential applications in gene therapy. (C) 2012 Elsevier Ireland Ltd. All rights reserved.

Therapeutic use of a cationic antimicrobial peptide from the spider Acanthoscurria gomesiana in the control of experimental candidiasis

Background: Antimicrobial peptides are present in animals, plants and microorganisms and play a fundamental role in the innate immune response. Gomesin is a cationic antimicrobial peptide purified from haemocytes of the spider Acanthoscurria gomesiana. It has a broad-spectrum of activity against bacteria, fungi, protozoa and tumour cells. Candida albicans is a commensal yeast that is part of the human microbiota. However, in immunocompromised patients, this fungus may cause skin, mucosal or systemic infections. The typical treatment for this mycosis comprises three major categories of antifungal drugs: polyenes, azoles and echinocandins; however cases of resistance to these drugs are frequently reported. With the emergence of microorganisms that are resistant to conventional antibiotics, the development of alternative treatments for candidiasis is important. In this study, we evaluate the efficacy of gomesin treatment on disseminated and vaginal candidiasis as well as its toxicity and biodistribution. Results: Treatment with gomesin effectively reduced Candida albicans in the kidneys, spleen, liver and vagina of infected mice. The biodistribution of gomesin labelled with technetium-99 m showed that the peptide is captured in the kidneys, spleen and liver. Enhanced production of TNF-alpha, IFN-gamma and IL-6 was detected in infected mice treated with gomesin, suggesting an immunomodulatory activity. Moreover, immunosuppressed and C. albicans-infected mice showed an increase in survival after treatment with gomesin and fluconazole. Systemic administration of gomesin was also not toxic to the mice Conclusions: Gomesin proved to be effective against experimental Candida albicans infection. It can be used as an alternative therapy for candidiasis, either alone or in combination with fluconazole. Gomesin's mechanism is not fully understood, but we hypothesise that the peptide acts through the permeabilisation of the yeast membrane leading to death and/or releasing the yeast antigens that trigger the host immune response against infection. Therefore, data presented in this study reinforces the potential of gomesin as a therapeutic antifungal agent in both humans and animals.

Stable assemblies of cationic bilayer fragments and CpG oligonucleotide with enhanced immunoadjuvant activity in vivo

The cationic lipid dioctadecyldimethylammonium bromide (DODAB) and the CpG oligonucleotide (CpG) have been separately used as potent immunoadjuvants driving Th1 responses. Here DODAB bilayer fragments (BF) and CpG (5 -TTGACGTTCG-3) assemblies have their physical properties and immunoadjuvant activity determined using ovalbumin (OVA) as a model antigen. At 0.1 mg/mL OVA, the dependence of DODAB BF/OVA size and zeta-potential on time and [DODAB] establishes 0.1 mMDODAB as suitable for obtaining stable and cationic DODAB BF/OVA assemblies. At 0.1 mMDODAB, 0.1 mg/mL OVA and 0.006 mMCpG, the zeta-potential is zero. At [CpG]>0.006 mM, good colloidal stability for the anionic assemblies is due to charge overcompensation. At 0.020 mM CpG, these DODAB BF/OVA/CpG assemblies are highly effective in vivo generating responses similar to those elicited by the stable and cationic DODAB BF/OVA. The anti-OVA DTH reaction and the secretion of IFN-gamma and IL-12 are 6, 42 and 9 times larger for the DODAB BF/OVA/CpG-immunized mice than the same responses by OVA-immunized mice, respectively. This work shows for the first time that charge of small assemblies is not important to determine the immune response. (C) 2011 Elsevier B. V. All rights reserved.

Photodynamic inactivation of biofilms formed by Candida spp., Trichosporon mucoides, and Kodamaea ohmeri by cationic nanoemulsion of zinc 2,9,16,23-tetrakis(phenylthio)-29H, 31H-phthalocyanine (ZnPc)

The biofilms formed by opportunistic yeasts serve as a persistent reservoir of infection and impair the treatment of fungal diseases. The aim of this study was to evaluate photodynamic inactivation (PDI) of biofilms formed by Candida spp. and the emerging pathogens Trichosporon mucoides and Kodamaea ohmeri by a cationic nanoemulsion of zinc 2,9,16,23-tetrakis(phenylthio)-29H,31H-phthalocyanine (ZnPc). Biofilms formed by yeasts after 48 h in the bottom of 96-well microtiter plates were treated with the photosensitizer (ZnPc) and a GaAlAs laser (26.3 J cm(-2)). The biofilm cells were scraped off the well wall, homogenized, and seeded onto Sabouraud dextrose agar plates that were then incubated at 37A degrees C for 48 h. Efficient PDI of biofilms was verified by counting colony-forming units (CFU/ml), and the data were submitted to analysis of variance and the Tukey test (p < 0.05). All biofilms studied were susceptible to PDI with statistically significant differences. The strains of Candida genus were more resistant to PDI than emerging pathogens T. mucoides and K. ohmeri. A mean reduction of 0.45 log was achieved for Candida spp. biofilms, and a reduction of 0.85 and 0.84, were achieved for biofilms formed by T. mucoides and K. ohmeri, respectively. Therefore, PDI by treatment with nanostructured formulations cationic zinc 2,9,16,23- tetrakis (phenylthio)- 29H, 31H- phthalocyanine (ZnPc) and a laser reduced the number of cells in the biofilms formed by strains of C. albicans and non-Candida albicans as well the emerging pathogens T. mucoides and K. ohmeri.

Development of Cationic Solid Lipid Nanoparticles with Factorial Design-Based Studies for Topical Administration of Doxorubicin

Topical chemotherapy using doxorubicin, a powerful anticancer drug, can be used as an alternative with reduced systemic toxicity when treating skin cancer. The aim of the present work was to use factorial design-based studies to develop cationic solid lipid nanoparticles containing doxorubicin; further investigations into the influence of these particles on the drug's cytotoxicity and cellular uptake in B16F10 murine melanoma cells were performed. A 3(2) full factorial design was applied for two different lipid phases; one phase used stearic acid and the other used a 1:2 mixture of stearic acid and glyceryl behenate. The two factors investigated included the ratio between the lipid and the water phase and the ratio between the surfactant (poloxamer) and the co-surfactant (cetylpyridinium chloride). It was observed that the studied factors did not affect the mean diameter or the polydispersity of the obtained nanoparticles; however, they did significantly affect the zeta potential values. Optimised formulations with particle sizes ranging from 251 to 306 nm and positive zeta potentials were selected for doxorubicin incorporation. High entrapment efficiencies were achieved (97%) in formulations with higher amounts of stearic acid, suggesting that cationic charges on doxorubicin molecules may interact with the negative charges in stearic acid. Melanoma culture cell experiments showed that cationic solid lipid nanoparticles without drug were not cytotoxic to melanoma cells. The encapsulation of doxorubicin significantly increased cytotoxicity, indicating the potential of these nanoparticles for the treatment of skin cancer.

beta-ZnMoO4 microcrystals synthesized by the surfactant-assisted hydrothermal method: Growth process and photoluminescence properties

In this communication, we investigate the effect of different surfactants: cetyltrimethylammonium bromide (CTAB), sodium dodecyl sulfate (SDS) and polyvinylpyrrolidone (PVP-K40) on the growth process of zinc molybdate (beta-ZnMoO4) microcrystals synthesized under hydrothermal conditions at 140 degrees C for 8 h. These microcrystals were characterized by X-ray diffraction (XRD), field emission scanning electron microscopy (FE-SEM), and photoluminescence (PL) measurements. XRD patterns proved that these crystals are monophasic and present a wolframite-type monoclinic structure. FE-SEM images revealed that the surfactants modified the crystal shapes, suggesting the occurrence of distinct crystal growth processes. The CTAB cationic surfactant promotes the hindrance of small nuclei that leads to the formation of rectangle-like crystals, SDS anionic surfactant induces a growth of irregular hexagons with several porous due to considerable size effect of counter-ions on the crystal facets, PVP-K40 non-ionic surfactant allows a reduction in size and thickness of plate-like crystals, while without surfactants have the formation of irregular plate-like crystals. Finally, the PL properties of beta-ZnMoO4 microcrystals were explained by means of different shape/size, surface defects and order-disorder into lattice. (C) 2011 Elsevier B.V. All rights reserved.

Effect of sorbitan-based surfactants on glass transition temperature of cellulose esters

Thermal behavior of mixtures composed of cellulose acetate butyrate (CAB), carboxymethylcellulose acetate butyrate (CMCAB), or cellulose acetate phthalate (CAPh), and sorbitan-based surfactants was investigated as a function of mixture composition by means of differential scanning calorimetry (DSC). Surfactants with three different alkyl chain lengths, namely, polyoxyethylenesorbitan monolaurate (Tween 20), polyoxyethylenesorbitan monopalmitate (Tween 40), and polyoxyethylene sorbitan monostearate (Tween 60) were chosen. DSC measurements revealed that Tween 20, 40, and 60 act as plasticizers for CAB, CMCAB, and CAPh (except for Tween 60), leading to a dramatic reduction of glass transition temperature (T-g). The dependence of experimental T-g values on the mixture composition was compared with theoretical predictions using the Fox equation. Plasticization was strongly dependent on mixture composition, surfactant hydrophobic chain length, and type of cellulose ester functional group.

Rheological Measurements and Thermal Characterization of Lamellar Gel Phase Emulsions Developed with Cetearyl Alcohol/Nonionic Ethoxylated Surfactants

The aim of this research was to characterize emulsion systems (ES) containing nonionic ethoxylated surfactants by using rheological, microscopic, and thermogravimetric assays. Three formulations were developed: ES-1: 8.0% (w/w) oleth-20; ES-2: 4.0% (w/w) oleth-20/4.0% (w/w) steareth-21; and ES-3: 8.0% (w/w) steareth-21. The systems showed typical non-Newtonian pseudo-plastic behavior. The presence of a lamellar gel phase was observed for all systems, with ES-2 being more pronounced. Through thermogravimetry, the profiles of the three systems were found to be similar, consisting of two main events, the first one being characterized by loss of water and, beyond 110 degrees C, by loss of the oil phase.

Cationic nanoparticles for delivery of amphotericin B: preparation, characterization and activity in vitro

Abstract Background Particulate systems are well known to be able to deliver drugs with high efficiency and fewer adverse side effects, possibly by endocytosis of the drug carriers. On the other hand, cationic compounds and assemblies exhibit a general antimicrobial action. In this work, cationic nanoparticles built from drug, cationic lipid and polyelectrolytes are shown to be excellent and active carriers of amphotericin B against C. albicans. Results Assemblies of amphotericin B and cationic lipid at extreme drug to lipid molar ratios were wrapped by polyelectrolytes forming cationic nanoparticles of high colloid stability and fungicidal activity against Candida albicans. Experimental strategy involved dynamic light scattering for particle sizing, zeta-potential analysis, colloid stability, determination of AmB aggregation state by optical spectra and determination of activity against Candida albicans in vitro from cfu countings. Conclusion Novel and effective cationic particles delivered amphotericin B to C. albicans in vitro with optimal efficiency seldom achieved from drug, cationic lipid or cationic polyelectrolyte in separate. The multiple assembly of antibiotic, cationic lipid and cationic polyelctrolyte, consecutively nanostructured in each particle produced a strategical and effective attack against the fungus cells.

Comparison of physical properties of two exogenous surfactants: new parameter.

The pulmonary surfactant has essential physical properties for normal lung function. The most important property is the surface tension. In this work, it was evaluated the surface tension of two commercial exogenous surfactants used in surfactant replacement therapy, poractant alfa (Curosurf, Chiesi Farmaceuticals, Italy) and beractant (Survanta, Abbott Laboratories, USA) using new parameters. A Langmuir film balance (Minitrough, KSV Instruments, Finland) was used to measure surface tension of poractant alfa and beractant samples. For both samples, we prepared a solution of 1 mg/m dissolved in chloroform (100π`), which was applied over a subphase of milli-Q water (175 ml) in the chamber of the balance. The chamber has two moving barriers that can change its surface area between a maximal value of 112.5 cm 2 , and a minimal value of 22.5 cm 2, defining a balance cycle. Each surfactant had its surface tension evaluated during 20 balance cycles for three times. Four quantities were calculated from the experiment: Minimum Surface Tension (MTS), defined as the surface tension at minimal surface area during the first cycle; Mean Work Cycle (MWC), defined as the mean hysteresis area of the measured surface tension curve of the last 16 balance cycles; Critical Active Surface Area in Compression (CASAC) or in Expansion (CASAE), defined as the maximal chamber area where the surfactant is active on the surface in compression or expansion. The t-test was applied to verify for statistical significance of the results. Comproved with the MST is the same reported in literature, the differences between MWC, CASAC, and CASAE were statistically significant (p<0.001). The MWC, CASAC and CASAE were higher for poractant alfa than for beractant. A higher MWC for poractant alfa means higher elastic recoil of the lung in comparison with beractant. Using a different methodology, our results showed that poractant alfa is probably more effective in a surfactant replacement therapy than beractant due the use of poractant alfa in relation to the use of beractant in preterm infants with Respiratory Distress Syndrome (RDS).

Mechanical Measurements Of Two Exogenous Surfactants Using A Langmuir Film Balance:A Comparative Study

Rationale: The primary function of surfactant is to reduce the surface tension at air-liquid interface. In this study, the surface tension behavior of two commercial surfactants, poractant alfa (ChiesiFarmaceuticals,ltaly) and beractant (Abbott Laboratories,USA), were evaluated,using new parameters. Methods: We used a Langmuir film balance (Minitrough,KSV lnstruments,Finland) to measure of surface tension of both poractant alfa and beractant samples. For both samples,we prepared a solution of 1 mg/mdl dissolved in chloroform. The solution (1uL) was applied over a subphase of milli-Q water (175 ml) in the chamber of the balance. The chamber has two moving barriers that can change its surface area between a maximum value of 112.5 cm2 anda minimum value of 22.5 cm2, defining a balance cycle.lhree sample's films were evaluated for each sample, during 20 balance cycles. Here quantify two new variables, which is the mean hysteresis area of the measured curve surface tension of the last 16 balance cycles,defined here as Mean Work Cycle (MWC), and the moment that the surfactant is active in the surface, this measure is defined here as Active Surface Area Critical in the compression (ASACC) and the expansion (ASACE). The test was applied to compare the statistical significance of the results.

Effect of interaction with micelles on the excited-state optical properties of zinc porphyrins and J-aggregates formation

This work reports on the photophysical properties of zinc porphyrins meso-tetrakis methylpyridiniumyl (Zn2+TMPyP) and meso-tetrakis sulfonatophenyl (Zn2+TPPS) in homogeneous aqueous solutions and in the presence of sodium dodecyl sulfate (SDS) and cetyltrimethyl ammonium bromide (CTAB) micelles. The excited-state dynamic was investigated with the Z-scan technique, UV-Vis absorption, and fluorescence spectroscopy. Photophysical parameters were obtained by analyzing the experimental data with a conventional five-energy-level diagram. The interaction of the charged side porphyrin groups with oppositely charged surfactants can reduce the electrostatic repulsion between porphyrin molecules leading to aggregation, which affected the porphyrin characteristics such as absorption cross-sections, lifetimes and quantum yields. The interaction between anionic ZnTPPS with cationic CTAB micelles induced the formation of porphyrin J-aggregates, while this effect was not observed in the interaction of ZnTMPyP with SDS micelles. This difference is, probably, due to the difference in electrostatic repulsion between the porphyrin molecules. The insights obtained by these results are important for the understanding of the photophysical behavior of porphyrins, regarding potential applications in pharmacokinetics as encapsulation of photosensitizer for drug delivery systems and in its interaction with cellular membrane.

Studies on the interaction of surfactants and neutral cyclodextrins by capillary electrophoresis. Application to chiral analysis

In the present study, mixed systems composed of SDS in the presence of neutral cyclodextrins were considered. Firstly, the effect of the CDs on the CMC of the surfactant was evaluated by CE experiments. Furthermore, a new CE approach based on electric current measurement was developed for the estimation of the stoichiometry as well as of the binding constants of SDS-CDs complexes. The results of these investigations were compared to those obtained with a different technique, electronic paramagnetic resonance (EPR). The obtained results suggested that methylated CDs, in particular (2,6-di-O-methyl)-beta-cyclodextrin (DM-beta-CD), strongly affect the micellization of SDS in comparison to the other studied CDs. This effect also paralleled the chiral CD-MEKC performance, as indicated by the enantioresolution of (+/-)-Catechin, which was firstly selected as a model compound representative of important chiral phytomarkers. Then a CD-MEKC system, composed of sodium dodecyl sulfate as surfactant (90 mM) and hydroxypropyl-beta-cyclodextrin (25 mM) as chiral selector, under acidic conditions (25 mM borate – phosphate buffer, pH 2.5) was applied to study the thermal epimerisation of epi-structured catechins, (-)-Epicatechin and (-)-Epigallocatechin, to non epi-structured (-)-Catechin and (-)-Gallocatechin. The latter compounds, being non-native molecules, were for the first time regarded as useful phytomarkers of tea sample degradation. The proposed method was applied to the analysis of more than twenty tea samples of different geographical origins (China, Japan, Ceylon), having undergone different storage conditions and manufacturing processes.