Iron-carbohydrate complexes treating iron anaemia: Understanding the nano-structure and interactions with proteins through orthogonal characterisation.

Intravenous (IV) iron-carbohydrate complexes are widely used nanoparticles (NPs) to treat iron deficiency anaemia, often associated with medical conditions such as chronic kidney disease, heart failure and various inflammatory conditions. Even though a plethora of physicochemical characterisation data and clinical studies are available for these products, evidence-based correlation between physicochemical properties of iron-carbohydrate complexes and clinical outcome has not fully been elucidated yet. Studies on other metal oxide NPs suggest that early interactions between NPs and blood upon IV injection are key to understanding how differences in physicochemical characteristics of iron-carbohydrate complexes cause variance in clinical outcomes. We therefore investigated the core-ligand structure of two clinically relevant iron-carbohydrate complexes, iron sucrose (IS) and ferric carboxymaltose (FCM), and their interactions with two structurally different human plasma proteins, human serum albumin (HSA) and fibrinogen, using a combination of cryo-scanning transmission electron microscopy (cryo-STEM), x-ray diffraction (XRD), small-angle x-ray scattering (SAXS) and small-angle neutron scattering (SANS). Using this orthogonal approach, we defined the nano-structure, individual building blocks and surface morphology for IS and FCM. Importantly, we revealed significant differences in the surface morphology of the iron-carbohydrate complexes. FCM shows a localised carbohydrate shell around its core, in contrast to IS, which is characterised by a diffuse and dynamic layer of carbohydrate ligand surrounding its core. We hypothesised that such differences in carbohydrate morphology determine the interaction between iron-carbohydrate complexes and proteins and therefore investigated the NPs in the presence of HSA and fibrinogen. Intriguingly, IS showed significant interaction with HSA and fibrinogen, forming NP-protein clusters, while FCM only showed significant interaction with fibrinogen. We postulate that these differences could influence bio-response of the two formulations and their clinical outcome. In conclusion, our study provides orthogonal characterisation of two clinically relevant iron-carbohydrate complexes and first hints at their interaction behaviour with proteins in the human bloodstream, setting a prerequisite towards complete understanding of the correlation between physicochemical properties and clinical outcome.

Stability Criterion for the Assembly of Core-Shell Lipid-Polymer-Nucleic Acid Nanoparticles.

Hybrid core-shell lipid-polycation-nucleic acid nanoparticles (LPNPs) provide unique delivery strategies for nonviral gene therapeutics. Since LPNPs consist of multiple components, involving different pairwise interactions between them, they are challenging to characterize and understand. Here, we propose a method based on fluorescence cross-correlation spectroscopy to elucidate the association between the three LPNP components. Through this lens, we demonstrate that cationic lipid shells (liposomes) do not displace polycations or DNA from the polycation-DNA cores (polyplexes). Hence, polyplexes and liposomes must be oppositely charged to associate into LPNPs. Furthermore, we identify the liposome:polyplex number ratio (ρN), which was hitherto an intangible quantity, as the primary parameter predicting stable LPNPs. We establish that ρN ≥ 1 ensures that every polyplex is enveloped by a liposome, thus avoiding coexisting oppositely charged species prone to aggregation.

A Versatile Nanocarrier-Cubosomes, Characterization, and Applications.

The impact of nanotechnology on the exponential growth of several research areas, particularly nanomedicine, is undeniable. The ability to deliver active molecules to the desired site could significantly improve the efficiency of medical treatments. One of the nanocarriers developed which has drawn researchers' attention are cubosomes, which are nanosized dispersions of lipid bicontinuous cubic phases in water, consisting of a lipidic interior and aqueous domains folded in a cubic lattice. They stand out due to their ability to incorporate hydrophobic, hydrophilic, and amphiphilic compounds, their tortuous internal configuration that provides a sustained release, and the capacity to protect and safely deliver molecules. Several approaches can be taken to prepare this structure, as well as different lipids like monoolein or phytantriol. This review paper describes the different methods to prepare nanocarriers. As it is known, the physicochemical properties of nanocarriers are very important, as they influence their pharmacokinetics and their ability to incorporate and deliver active molecules. Therefore, an extensive characterization is essential to obtain the desired effect. As a result, we have extensively described the most common techniques to characterize cubosomes, particularly nanocarriers. The exceptional properties of the cubosomes make them suitable to be used in several applications in the biomedical field, from cancer therapeutics to imaging, which will be described. Taking in consideration the outstanding properties of cubosomes, their application in several research fields is envisaged.

Cationic lipid-based formulations for encapsulation and delivery of anti-EFG1 2' OMethylRNA oligomer.

The effective protection and delivery of antisense oligomers to its site of action is a challenge without an optimal strategy. Some of the most promising approaches encompass the complexation of nucleic acids, which are anionic, with liposomes of fixed or ionizable cationic charge. Thus, the main purpose of this work was to study the complexation of cationic liposomes with anti-EFG1 2'OMe oligomers and evaluate the complex efficacy to control Candida albicans filamentation in vitro and in vivo using a Galleria mellonella model. To accomplish this, cationic dioleoyl-trimethylammoniumpropane (DOTAP) was mixed with three different neutral lipids dioleoyl-phosphocholine (DOPC), dioleoyl-phosphatidylethanolamine (DOPE) and monoolein (MO) and used as delivery vectors. Fluorescence Cross Correlation Spectroscopy measurements revealed a high association between antisense oligomers (ASO) and cationic liposomes confirming the formation of lipoplexes. In vitro, all cationic liposome-ASO complexes were able to release the anti-EFG1 2'OMe oligomers and consequently inhibit C. albicans filamentation up to 60% after 72 h. In vivo, from all formulations the DOTAP/DOPC 80/20 ρchg = 3 formulation proved to be the most effective, enhancing the G. mellonella survival by 40% within 48 h and by 25% after 72 h of infection. In this sense, our findings show that DOTAP-based lipoplexes are very good candidates for nano-carriers of anti-EFG1 2'OMe oligomers.

In Vitro CRISPR/Cas9 Transfection and Gene-Editing Mediated by Multivalent Cationic Liposome-DNA Complexes.

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated nuclease 9 (Cas9) gene-editing offers exciting new therapeutic possibilities for disease treatment with a genetic etiology such as cancer, cardiovascular, neuronal, and immune disorders. However, its clinical translation is being hampered by the lack of safe, versatile, and effective nonviral delivery systems. Herein we report on the preparation and application of two cationic liposome−DNA systems (i.e., lipoplexes) for CRISPR/Cas9 gene delivery. For that purpose, two types of cationic lipids are used (DOTAP, monovalent, and MVL5, multivalent with +5e nominal charge), along with three types of helper lipids (DOPC, DOPE, and monoolein (GMO)). We demonstrated that plasmids encoding Cas9 and single-guide RNA (sgRNA), which are typically hard to transfect due to their large size (>9 kb), can be successfully transfected into HEK 293T cells via MVL5-based lipoplexes. In contrast, DOTAP-based lipoplexes resulted in very low transfection rates. MVL5-based lipoplexes presented the ability to escape from lysosomes, which may explain the superior transfection efficiency. Regarding gene editing, MVL5-based lipoplexes achieved promising GFP knockout levels, reaching rates of knockout superior to 35% for charge ratios (+/−) of 10. Despite the knockout efficiency being comparable to that of Lipofectamine 3000® commercial reagent, the non-specific gene knockout is more pronounced in MVL5-based formulations, probably resulting from the considerable cytotoxicity of these formulations. Altogether, these results show that multivalent lipid-based lipoplexes are promising CRISPR/Cas9 plasmid delivery vehicles, which by further optimization and functionalization may become suitable in vivo delivery systems.

Cladonia verticillaris (lichen) indicates negative impacts derived from the combustion of biodiesel blends: an alert for the environmental management for biofuels use.

The use of biodiesel blends with petroleum diesel in vehicular engines demands the evaluation of the possible impacts and effects of the gases emitted from their combustion on the environment. Among studies on these questions, biomonitoring using lichens is a viable alternative, given their interactions with the elements dispersed in the atmosphere, as well as its sensitivity and capacity to retain contaminants. In this study, we analyzed the effects of gas emissions from the combustion of biodiesel mixture with petroleum diesel on Cladonia verticillaris thalli. Samples of the lichen (10 g) were exposed to the gases emitted by the exhaust of the generator engine during the combustion process of biodiesel mixtures to petroleum diesel (7% (B7), 10% (B10), 40% (B40), 50% (B50), and 70% (B70)). At 90 days after exposure, samples were analyzed for n-alkane profiles, thallus morphology, photosynthetic pigment contents, and secondary lichen metabolites (protocetraric and fumarprotocetraric acids). Sets B7 and B10 showed better resistance of the lichen to pollutants. Set B40 showed a high stress evidenced by the chain elongation of n-alkanes structure and high chlorophyll production, presenting high morphological damages when compared to the control sets, B7 and B10. The results showed significant reductions of n-alkanes profiles for mixtures with high concentrations of biodiesel (B50 and B70), as well as decreases in the chlorophyll content. These groups showed an increase in the synthesis of secondary metabolites, corroborating the hypothesis that high concentrations of biodiesel in the mixture with petroleum diesel have greater impacts on the lichen. Schematic model for demonstration of using the lichen Cladonia verticillaris as biomonitor of effects from gas emissions from the combustion of biodiesel blends with petroleum diesel by a stationary engine.

Sustained Release of a Streptococcus pneumoniae Endolysin from Liposomes for Potential Otitis Media Treatment.

Local delivery of antimicrobials for otitis media treatment would maximize therapeutic efficacy while minimizing side effects. However, drug transport across the tympanic membrane in the absence of a delivery system is challenging. In this study, the MSlys endolysin was encapsulated in deformable liposomes for a targeted treatment of S. pneumoniae, one of the most important causative agents of otitis media. MSlys was successfully encapsulated in liposomes composed of l-alpha-lecithin and sodium cholate (5:1) or l-alpha-lecithin and PEG2000 PE (10:1), with encapsulation efficiencies of about 35%. The PEGylated and sodium cholate liposomes showed, respectively, mean hydrodynamic diameters of 85 and 115 nm and polydispersity indices of 0.32 and 0.42, both being stable after storage at 4 °C for at least one year. Both liposomal formulations showed a sustained release of MSlys over 7 days. Cytotoxicity studies against fibroblast and keratinocyte cell lines revealed the biocompatible nature of both MSlys and MSlys-loaded liposomes. Additionally, the encapsulated MSlys showed prompt antipneumococcal activity against planktonic and biofilm S. pneumoniae, thus holding great potential for transtympanic treatment against S. pneumoniae otitis media.

Fluorescence cross-correlation spectroscopy as a valuable tool to characterize cationic liposome-DNA nanoparticle assembly.

The development of nonviral gene delivery vehicles for therapeutic applications requires methods capable of quantifying the association between the genes and their carrier counterparts. Here we investigate the potential of fluorescence cross-correlation spectroscopy (FCCS) to characterize and optimize the assembly of nonviral cationic liposome (CL)-DNA complexes based on a CL formulation consisting of the cationic lipid DOTAP and zwitterionic lipid DOPC. We use a DNA plasmid for lipoplex loading encoding the Oct4 gene, critically involved in reprogramming somatic cells into induced pluripotent stem cells. We demonstrate that FCCS is able to quantitatively determine the extent of the association between DNA and the liposomes and assess its loading capacity. We also establish that the cationic lipid fraction, being proportional to the liposome membrane charge density, as well as charge ratio between the CLs and anionic DNA play an important role in the degree of interaction between the liposomes and DNA.

Rhamnolipids-based nanostructured lipid carriers: Effect of lipid phase on physicochemical properties and stability.

In this work rhamnolipids were evaluated as surfactants for the production of nanostructured lipid carriers (NLCs). NLCs were produced by melt-emulsification using ultra-homogenisation followed by ultrasonication and different ratios of medium-chain-triglycerides and glycerol monostearate (lipid phase) were tested. NLCs presented sizes and polydispersity index values ranged between 97 and 120 nm and 0.20-0.26, respectively. Transmission electron microscopy observations confirmed the size and the spherical morphology of the NLCs. The thermal analysis and X-ray diffraction showed that the amount of solid lipid (glycerol monostearate) influences the melting, crystallisation and enthalpy of NLCs and their degree of crystallinity. Results showed that NLCs were more stable at 4 °C and the best formulation (1% of water phase, 0.05% of biosurfactant and solid:liquid ratio of 10:90) was stable for 30 days. This work showed the possibility of using rhamnolipids to produce NLCs and represent an important step for the development of lipid-based nanosystems using biosurfactants.

Lipid-Nucleic Acid Complexes: Physicochemical Aspects and Prospects for Cancer Treatment.

Cancer is an extremely complex disease, typically caused by mutations in cancer-critical genes. By delivering therapeutic nucleic acids (NAs) to patients, gene therapy offers the possibility to supplement, repair or silence such faulty genes or to stimulate their immune system to fight the disease. While the challenges of gene therapy for cancer are significant, the latter approach (a type of immunotherapy) starts showing promising results in early-stage clinical trials. One important advantage of NA-based cancer therapies over synthetic drugs and protein treatments is the prospect of a more universal approach to designing therapies. Designing NAs with different sequences, for different targets, can be achieved by using the same technologies. This versatility and scalability of NA drug design and production on demand open the way for more efficient, affordable and personalized cancer treatments in the future. However, the delivery of exogenous therapeutic NAs into the patients' targeted cells is also challenging. Membrane-type lipids exhibiting permanent or transient cationic character have been shown to associate with NAs (anionic), forming nanosized lipid-NA complexes. These complexes form a wide variety of nanostructures, depending on the global formulation composition and properties of the lipids and NAs. Importantly, these different lipid-NA nanostructures interact with cells via different mechanisms and their therapeutic potential can be optimized to promising levels in vitro. The complexes are also highly customizable in terms of surface charge and functionalization to allow a wide range of targeting and smart-release properties. Most importantly, these synthetic particles offer possibilities for scaling-up and affordability for the population at large. Hence, the versatility and scalability of these particles seem ideal to accommodate the versatility that NA therapies offer. While in vivo efficiency of lipid-NA complexes is still poor in most cases, the advances achieved in the last three decades are significant and very recently a lipid-based gene therapy medicine was approved for the first time (for treatment of hereditary transthyretin amyloidosis). Although the path to achieve efficient NA-delivery in cancer therapy is still long and tenuous, these advances set a new hope for more treatments in the future. In this review, we attempt to cover the most important biophysical and physicochemical aspects of non-viral lipid-based gene therapy formulations, with a perspective on future cancer treatments in mind.

Rapidly dissolving microneedles for the delivery of cubosome-like liquid crystalline nanoparticles with sustained release of rapamycin.

In this study, we developed a system for the transdermal delivery and controlled release of the hydrophobic immunosuppressive drug rapamycin, foreseeing an application in psoriasis treatment. To do so, rapamycin was encapsulated in phytantriol-based cubosome-like liquid crystalline nanoparticles stabilized with pluronic F127. The final mass percent composition of the lipid nanoparticles was 0.25% phytantriol, 0.1% pluronic F127, 4.75% ethanol and 94.9% water. These particles showed a rapamycin encapsulation efficiency above 95% and a sustained in vitrodrug release profile throughout 14 days. Subsequently the rapamycin-carrying particles were incorporated into rapidly dissolving microneedle patches composed of a polymeric matrix of poly(vinylpyrrolidone) and poly(vinyl alcohol). Confocal microscopy allowed to infer the preferential distribution of the cubosome-like particles at the tip and baseplate of the microneedles. The fabricated microneedles showed successful piercing and deposition of the loaded cubosome-like particles on a skin-mimicking agarose gel. Finally, the rapamycin-loaded cubosome-like particles showed antiproliferative activity in natural killer cells in vitro. The results here presented show the potential of the developed system to deliver cubosome-like particles into the skin and promote the sustained release of rapamycin in the context of immunomodulation.

In Vitro Evaluation of Lipopolyplexes for Gene Transfection: Comparing 2D, 3D and Microdroplet-Enabled Cell Culture.

Complexes combining nucleic acids with lipids and polymers (lipopolyplexes) show great promise for gene therapy since they enable compositional, physical and functional versatility to be optimized for therapeutic efficiency. When developing lipopolyplexes for gene delivery, one of the first evaluations performed is an in vitro transfection efficiency experiment. Many different in vitro models can be used, and the effect of the model on the experiment outcome has not been thoroughly studied. The objective of this work was to compare the insights obtained from three different in vitro models, as well as the potential limitations associated with each of them. We have prepared a series of lipopolyplex formulations with three different cationic polymers (poly-l-lysine, bioreducible poly-l-lysine and polyethyleneimine), and assessed their in vitro biological performance in 2D monolayer cell culture, 3D spheroid culture and microdroplet-based single-cell culture. Lipopolyplexes from different polymers presented varying degrees of transfection efficiency in all models. The best-performing formulation in 2D culture was the polyethyleneimine lipopolyplex, while lipoplexes prepared with bioreducible poly-l-lysine were the only ones achieving any transfection in microdroplet-enabled cell culture. None of the prepared formulations achieved significant gene transfection in 3D culture. All of the prepared formulations were well tolerated by cells in 2D culture, while at least one formulation (poly-l-lysine polyplex) delayed 3D spheroid growth. These results highlight the need for selecting the appropriate in vitro model depending on the intended application.

The Impact of Sublethal Concentrations of Treated Leachate on Nile tilápia (Oreochromis niloticus).

The objective of this study was to evaluate the impact of sublethal concentrations of treated landfill leachate on Oreochromis niloticus individuals after exposure for 96 h, by assessing biochemical, genotoxic and immunologic biomarkers. Among biochemical biomarkers (activities of ALT, AST and GST enzymes), the treated landfill leachate did not cause significant alterations on O. niloticus and did not significantly affect leukocytes used as an immunologic biomarker. On the other hand, treated leachate induced genotoxic damages, since an increase in erythrocytic micronuclei and in DNA damage (comet assay) were observed in fish exposed to all treatment (2, 4 and 6 mL L-1). Acute toxicity of treated leachate in O. niloticus caused only genotoxic changes in blood cells, showing that micronuclei and comet assay, together, are effective biomarkers in determining the acute toxicity of treated leachate in aquatic environments. This work also shows that leachate, although treated, caused some damages to O. niloticus, which indicates the employed treatment was not efficient in eliminating all genotoxic substances from the leachate.

Enhanced magnetic microcytometer with 3D flow focusing for cell enumeration.

We report the design and characterization of a lateral and vertical hydrodynamic focusing feature for whole cell detection on a miniaturized flow cytometer. The developed system, based on magnetic sensing, incorporates spin valve sensors on the bottom of the microfluidic channels that detect cells labeled with magnetic beads. An adaptable 3D hydrodynamic focusing system was developed that pushes labeled cells towards the bottom of the microchannel, closer to the sensors, allowing increased signal amplitude for cells labeled with magnetic beads and enhanced discrimination of labeled cells. Fluorescence microscopy indicates that the lateral and vertical hydrodynamic focusing effect was adequately implemented, consistent with simulation predictions. The sensitivity of the system to detect labeled cells was improved by at least two-fold. By estimating the coverage of magnetic beads on cells, the signal from labeled cells could be predicted using a mathematical model, which also demonstrated the sensitivity of the signal to the height of the cells relative to the sensor. The system is versatile allowing interchangeable flow rates for cells with different diameters.

Unravelling a Mechanism of Action for a Cecropin A-Melittin Hybrid Antimicrobial Peptide: The Induced Formation of Multilamellar Lipid Stacks.

An understanding of the mechanism of action of antimicrobial peptides is fundamental to the development of new and more active antibiotics. In the present work, we use a wide range of techniques (SANS, SAXD, DSC, ITC, CD, and confocal and electron microscopy) in order to fully characterize the interaction of a cecropin A-melittin hybrid antimicrobial peptide, CA(1-7)M(2-9), of known antimicrobial activity, with a bacterial model membrane of POPE/POPG in an effort to unravel its mechanism of action. We found that CA(1-7)M(2-9) disrupts the vesicles, inducing membrane condensation and forming an onionlike structure of multilamellar stacks, held together by the intercalated peptides. SANS and SAXD revealed changes induced by the peptide in the lipid bilayer thickness and the bilayer stiffening in a tightly packed liquid-crystalline lamellar phase. The analysis of the observed abrupt changes in the repeat distance upon the phase transition to the gel state suggests the formation of an Lγ phase. To the extent of our knowledge, this is the first time that the Lγ phase is identified as part of the mechanism of action of antimicrobial peptides. The energetics of interaction depends on temperature, and ITC results indicate that CA(1-7)M(2-9) interacts with the outer leaflet. This further supports the idea of a surface interaction that leads to membrane condensation and not to pore formation. As a result, we propose that this peptide exerts its antimicrobial action against bacteria through extensive membrane disruption that leads to cell death.

SAXS on a chip: from dynamics of phase transitions to alignment phenomena at interfaces studied with microfluidic devices.

The field of microfluidics offers attractive possibilities to perform novel experiments that are difficult (or even impossible) to perform using conventional bulk and surface-based methods. Such attractiveness comes from several important aspects inherent to these miniaturized devices. First, the flow of fluids under submillimeter confinement typically leads to a drop of inertial forces, meaning that turbulence is practically suppressed. This leads to predictable and controllable flow profiles, along with well-defined chemical gradients and stress fields that can be used for controlled mixing and actuation on the micro and nanoscale. Secondly, intricate microfluidic device designs can be fabricated using cleanroom standard procedures. Such intricate geometries can take diverse forms, designed by researchers to perform complex tasks, that require exquisite control of flow of several components and gradients, or to mimic real world examples, facilitating the establishment of more realistic models. Thirdly, microfluidic devices are usually compatible with in situ or integrated characterization methods that allow constant real-time monitoring of the processes occurring inside the microchannels. This is very different from typical bulk-based methods, where usually one can only observe the final result, or otherwise, take quick snapshots of the evolving process or take aliquots to be analyzed separately. Altogether, these characteristics inherent to microfluidic devices provide researchers with a set of tools that allow not only exquisite control and manipulation of materials at the micro and nanoscale, but also observation of these effects. In this review, we will focus on the use and prospects of combining microfluidic devices with in situ small-angle X-ray scattering (and related techniques such as small-angle neutron scattering and X-ray photon correlation spectroscopy), and their enormous potential for physical-chemical research, mainly in self-assembly and phase-transitions, and surface characterization.

Fluorescence microscopy colocalization of lipid-nucleic acid nanoparticles with wildtype and mutant Rab5-GFP: A platform for investigating early endosomal events.

Endosomal entrapment is known to be a major bottleneck to successful cytoplasmic delivery of nucleic acids (NAs) using cationic liposome-NA nanoparticles (NPs). Quantitative measurements of distributions of NPs within early endosomes (EEs) have proven difficult due to the sub-resolution size and short lifetime of wildtype EEs. In this study we used Rab5-GFP, a member of the large family of GTPases which cycles between the plasma membrane and early endosomes, to fluorescently label early endosomes. Using fluorescence microscopy and quantitative image analysis of cells expressing Rab5-GFP, we found that at early time points (t<1h), only a fraction (≈35%) of RGD-tagged NPs (which target cell surface integrins) colocalize with wildtype EEs, independent of the NP's membrane charge density. In comparison, a GTP-hydrolysis deficient mutant, Rab5-Q79L, which extends the size and lifetime of EEs yielding giant early endosomes (GEEs), enabled us to resolve and localize individual NPs found within the GEE lumen. Remarkably, nearly all intracellular NPs are found to be trapped within GEEs implying little or no escape at early time points. The observed small degree of colocalization of NPs and wildtype Rab5 is consistent with recycling of Rab5-GDP to the plasma membrane and not indicative of NP escape from EEs. Taken together, our results show that endosomal escape of PEGylated nanoparticles occurs downstream of EEs i.e., from late endosomes/lysosomes. Our studies also suggest that Rab5-Q79L could be used in a robust imaging assay which allows for direct visualization of NP interactions with the luminal membrane of early endosomes.

Nematic director reorientation at solid and liquid interfaces under flow: SAXS studies in a microfluidic device.

In this work we investigate the interplay between flow and boundary condition effects on the orientation field of a thermotropic nematic liquid crystal under flow and confinement in a microfluidic device. Two types of experiments were performed using synchrotron small-angle X-ray-scattering (SAXS). In the first, a nematic liquid crystal flows through a square-channel cross section at varying flow rates, while the nematic director orientation projected onto the velocity/velocity gradient plane is measured using a 2D detector. At moderate-to-high flow rates, the nematic director is predominantly aligned in the flow direction, but with a small tilt angle of ∼±11° in the velocity gradient direction. The director tilt angle is constant throughout most of the channel width but switches sign when crossing the center of the channel, in agreement with the Ericksen-Leslie-Parodi (ELP) theory. At low flow rates, boundary conditions begin to dominate, and a flow profile resembling the escaped radial director configuration is observed, where the director is seen to vary more smoothly from the edges (with homeotropic alignment) to the center of the channel. In the second experiment, hydrodynamic focusing is employed to confine the nematic phase into a sheet of liquid sandwiched between two layers of Triton X-100 aqueous solutions. The average nematic director orientation shifts to some extent from the flow direction toward the liquid boundaries, although it remains unclear if one tilt angle is dominant through most of the nematic sheet (with abrupt jumps near the boundaries) or if the tilt angle varies smoothly between two extreme values (∼90 and 0°). The technique presented here could be applied to perform high-throughput measurements for assessing the influence of different surfactants on the orientation of nematic phases and may lead to further improvements in areas such as boundary lubrication and clarifying the nature of defect structures in LC displays.

Acute copper toxicity in juvenile fat snook Centropomus parallelus (Teleostei: Centropomidae) in sea water

Three experiments were designed to assess the accumulation and acute toxicity of copper (Cu) in juvenile fat snook Centropomus parallelus. The first experiment was performed to determine the 96-h lethal concentration (LC50) of Cu. The second experiment was designed to assess the effects of sublethal concentrations of Cu (0.47 and 0.94 mg/L), while the third one allowed us to test the recovery capacity of fish exposed to the sublethal concentrations Cu and kept in sea water without Cu addition. The LC50 value for Cu was found to be 1.88 mg/L Cu. Fish exposed to the sublethal concentrations of Cu showed a significant accumulation of Cu in gills at 96 h respect to the control ones (0.43 µg/g Cu). No significant difference was observed in the accumulation of Cu in gills between fish exposed to 0.47 mg/L (1.09 µg/g Cu) and 0.94 mg/L (1.26 µg/g Cu). Exposure (24 and 96 h) to the sublethal concentrations of Cu tested induced DNA damage in the erythrocytes. The results show that acute exposure to sublethal concentrations induces Cu accumulation and DNA damage in fish, these effects being recovered after 240 h in sea water without Cu addition.

Três experimentos foram realizados para avaliar o acúmulo e toxicidade aguda do cobre (Cu) em juvenis de robalo-peva Centropomus parallelus. O primeiro experimento foi realizado para determinar a concentração letal (96h-CL50) de Cu. O segundo experimento foi realizado para avaliar os efeitos de concentrações subletais de Cu (0,47 e 0,94 mg/L), enquanto o terceiro permitiu testar a capacidade de recuperação dos peixes expostos a concentrações subletais do Cu e posteriormente mantidos em água do mar sem acréscimo de Cu. O valor de LC50 encontrado para o Cu foi de 1,88 mg/L. Os peixes expostos as concentrações subletais de Cu mostraram um acúmulo significativo nas brânquias em relação ao controle em 96 h de exposição (0,43 µg/g Cu). Nenhuma diferença significativa foi observada entre os peixes expostos a 0,47 mg/L de Cu (1,09 µg/g) e 0,94 mg/L de Cu (1,26 µg/g). A exposição (24 e 96 h) para as concentrações subletais de Cu induziram danos no DNA. Os resultados mostram que a exposição aguda a concentrações subletais induz o acúmulo de Cu e danos ao DNA nas brânquias dos peixes, onde estes efeitos são recuperados após 240 h em água do mar sem adição de Cu.

Uptake and transfection efficiency of PEGylated cationic liposome-DNA complexes with and without RGD-tagging.

Steric stabilization of cationic liposome-DNA (CL-DNA) complexes is required for in vivo applications such as gene therapy. PEGylation (PEG: poly(ethylene glycol)) of CL-DNA complexes by addition of PEG2000-lipids yields sterically stabilized nanoparticles but strongly reduces their gene delivery efficacy. PEGylation-induced weakening of the electrostatic binding of CL-DNA nanoparticles to cells (leading to reduced uptake) has been considered as a possible cause, but experimental results have been ambiguous. Using quantitative live-cell imaging in vitro, we have investigated cell attachment and uptake of PEGylated CL-DNA nanoparticles with and without a custom synthesized RGD-peptide grafted to the distal ends of PEG2000-lipids. The RGD-tagged nanoparticles exhibit strongly increased cellular attachment as well as uptake compared to nanoparticles without grafted peptide. Transfection efficiency of RGD-tagged PEGylated CL-DNA NPs increases by about an order of magnitude between NPs with low and high membrane charge density (σM; the average charge per unit area of the membrane; controlled by the molar ratio of cationic to neutral lipid), even though imaging data show that uptake of RGD-tagged particles is only slightly enhanced by high σM. This suggests that endosomal escape and, as a result, transfection efficiency of RGD-tagged NPs is facilitated by high σM. We present a model describing the interactions between PEGylated CL-DNA nanoparticles and the anionic cell membrane which shows how the PEG grafting density and membrane charge density affect adhesion of nanoparticles to the cell surface.