DNA gel particles: an overview

A general understanding of interactions between DNA and oppositely charged compounds forms the basis for developing novel DNA-based materials, including gel particles. The association strength, which is altered by varying the chemical structure of the cationic cosolute, determines the spatial homogeneity of the gelation process, creating DNA reservoir devices and DNA matrix devices that can be designed to release either single- (ssDNA) or double-stranded (dsDNA) DNA. This review covers recent developments on the topic of DNA gel particles formed in water–water emulsion-type interfaces. The degree of DNA entrapment, particle morphology, swelling/dissolution behavior and DNA release responses are discussed as functions of the nature of the cationic agent used. On the basis of designing DNA gel particles for therapeutic purposes, recent studies on the determination of the surface hydrophobicity and the hemolytic and the cytotoxic assessments of the obtained DNA gel particles have been also reported

Mechanisms underlying cytotoxicity induced by engineered nanomaterials: a review of in vitro studies

Engineered nanomaterials are emerging functional materials with technologically interesting properties and a wide range of promising applications, such as drug delivery devices, medical imaging and diagnostics, and various other industrial products. However, concerns have been expressed about the risks of such materials and whether they can cause adverse effects. Studies of the potential hazards of nanomaterials have been widely performed using cell models and a range of in vitro approaches. In the present review, we provide a comprehensive and critical literature overview on current in vitro toxicity test methods that have been applied to determine the mechanisms underlying the cytotoxic effects induced by the nanostructures. The small size, surface charge, hydrophobicity and high adsorption capacity of nanomaterial allow for specific interactions within cell membrane and subcellular organelles, which in turn could lead to cytotoxicity through a range of different mechanisms. Finally, aggregating the given information on the relationships of nanomaterial cytotoxic responses with an understanding of its structure and physicochemical properties may promote the design of biologically safe nanostructures

Erythrocytes and cell line-based assays to evaluate the cytoprotective activity of antioxidant components obtained from natural sources

Oxidative stress can damage cellular components including DNA, proteins or lipids, and may cause several skin diseases. To protect from this damage and addressing consumer's appeal to natural products, antioxidants obtained from algal and vegetal extracts are being proposed as antioxidants to be incorporated into formulations. Thus, the development of reliable, quick and economic in vitro methods to study the cytoactivity of these products is a meaningful requirement. A combination of erythrocyte and cell line-based assays was performed on two extracts from Sargassum muticum, one from Ulva lactuca, and one from Castanea sativa. Antioxidant properties were assessed in erythrocytes by the TBARS and AAPH assays, and cytotoxicity and antioxidant cytoprotection were assessed in HaCaT and 3T3 cells by the MTT assay. The extracts showed no antioxidant activity on the TBARS assay, whereas their antioxidant capacity in the AAPH assay was demonstrated. On the cytotoxicity assays, extracts showed low toxicity, with IC50 values higher than 200 µg/mL. C. sativa extract showed the most favourable antioxidant properties on the antioxidant cytoprotection assays; while S. muticum and U. lactuca extracts showed a low antioxidant activity. This battery of methods was useful to characterize the biological antioxidant properties of these natural extracts

Mixed Protein-DNA Gel Particles for DNA Delivery: Role of Protein Composition and Preparation Method on Biocompatibility

Mixtures of two cationic proteins were used to prepare protein-DNA gel particles, employing associative phase separation and interfacial diffusion (Morán et al., 2009a). By mixing the two proteins, we have obtained particles that displayed higher loading efficiency and loading capacity values than those obtained in single-protein systems. However, nothing is known about the adverse effects on haemocompatibility and cytotoxicity of these protein-DNA gel particles. Here, we examined the interaction of protein-DNA gel particles obtained by two different preparation methods, and their components, with red blood cells and established cells. From a haemolytic point of view, these protein-DNA gel particles were demonstrated to be promising long-term blood-contacting medical devices. Safety evaluation with the established cell lines revealed that, in comparison with proteins in solution, the cytotoxicity was reduced when administered in the protein-DNA systems. In comparison with large-sized particles, the cytotoxic responses of small-sized protein-DNA gel particles showed to be strongly dependent of both the protein composition and the cell line being the tumour cell line HeLa more sensitive to the deleterious effects of the mixed protein-based particles. The observed trends in haemolysis and cell viabilities were in agreement with the degree of complexation values obtained for the protein-DNA gel particles prepared by both preparation methods

DNA gel particles: An overview

A general understanding of interactions between DNA and oppositely charged compounds forms the basis for developing novel DNA-based materials, including gel particles. The association strength, which is altered by varying the chemical structure of the cationic cosolute, determines the spatial homogeneity of the gelation process, creating DNA reservoir devices and DNA matrix devices that can be designed to release either single- (ssDNA) or double-stranded (dsDNA). This review covers recent developments on the topic of DNA gel particles formed in water-water emulsion-type interfaces. The degree of DNA entrapment, particle morphology, swelling/dissolution behaviour and DNA release responses are discussed as a function of the nature of the cationic agent used. On the basis of designing DNA gel particles for therapeutic purposes, recent studies on the determination of the surface hydrophobicity, the haemolytic and the cytotoxic assessments of the obtained DNA gel particles have been also reported

The underlying process of early ecological and genetic differentiation in a facultative mutualistic Sinorhizobium meliloti population

The question of how genotypic and ecological units arise and spread in natural microbial populations remains controversial in the field of evolutionary biology. Here, we investigated the early stages of ecological and genetic differentiation in a highly clonal sympatric Sinorhizobium meliloti population. Whole-genome sequencing revealed that a large DNA region of the symbiotic plasmid pSymB was replaced in some isolates with a similar synteny block carrying densely clustered SNPs and displaying gene acquisition and loss. Two different versions of this genomic island of differentiation (GID) generated by multiple genetic exchanges over time appear to have arisen recently, through recombination in a particular clade within this population. In addition, these isolates display resistance to phages from the same geographic region, probably due to the modification of surface components by the acquired genes. Our results suggest that an underlying process of early ecological and genetic differentiation in S. meliloti is primarily triggered by acquisition of genes that confer resistance to soil phages within particular large genomic DNA regions prone to recombination.España, Ministerio de Economía y Competitividad BIO 2014-51953-

Nanoparticles incorporating pH-responsive surfactants as a viable approach to improve the intracellular drug delivery

The pH-responsive delivery systems have brought newadvances in the field of functional nanodevices and might allow more accurate and controllable delivery of specific cargoes, which is expected to result in promising applications in different clinical therapies. Here we describe a family of chitosan TPP (tripolyphosphate) nanoparticles (NPs) for intracellular drug delivery, which were designed using two pH-sensitive amino acid-based surfactants fromthe family Nα,Nε-dioctanoyl lysine as bioactive compounds. Lowand mediummolecularweight chitosan (LMW-CS and MMW-CS, respectively) were used for NP preparation, and it was observed that the size distribution for NPs with LMW-CS were smaller (~168 nm) than that for NPs prepared with MMW-CS (~310 nm). Hemolysis assay demonstrated the pH-dependent biomembrane disruptional capability of the constructed NPs. The nanostructures incorporating the surfactants cause negligible membrane permeabilization at pH 7.4. However, at acidic pH, prevailing in endosomes, membrane-destabilizing activity in an erythrocyte lysis assay became evident. When pH decreased to 6.6 and 5.4, hemolytic capability of chitosan NPs increased along with the raise of concentration. Furthermore, studies with cell culture showed that these pH-responsive NPs displayed low cytotoxic effects against 3T3 fibroblasts. The influence of chitosan molecular weight, chitosan to TPP weight ratio, nanoparticle size and nature of the surfactant counterion on the membrane-disruptive properties of nanoparticleswas discussed in detail. Altogether, the results achieved here showed that by inserting the lysine-based amphiphiles into chitosan NPs, pH-sensitive membranolytic and potentially endosomolytic nanocarriers were developed, which, therefore, demonstrated ideal feasibility for intracellular drug delivery

Multifunctional PLGA nanoparticles combining transferrin-targetability and pH-stimuli sensitivity enhanced doxorubicin intracellular delivery and in vitro antineoplastic activity in MDR tumor cells

Targeted delivery aims to enhance cellular uptake and improve therapeutic outcome with higher disease specificity. The expression of transferrin receptor (TfR) is upregulated on tumor cells, which make the protein Tf and its receptor vastly relevant when applied to targeting strategies. Here, we proposed Tf-decorated pH-sensitive PLGA nanoparticles containing the chemosensitizer poloxamer as a carrier for doxorubicin delivery to tumor cells (Tf-DOX-PLGA-NPs), aiming at alleviating multidrug resistance (MDR). We performed a range of in vitro studies to assess whether targeted NPs have the ability to improve DOX antitumor potential on resistant NCI/ADR-RES cells. All evaluations of the Tf-decorated NPs were performed comparatively to the nontargeted counterparts, aiming to evidence the real role of NP surface functionalization, along with the benefits of pH-sensitivity and poloxamer, in the improvement of antiproliferative activity and reversal of MDR. Tf-DOX-PLGA-NPs induced higher number of apoptotic events and ROS generation, along with cell cycle arrest. Moreover, they were efficiently internalized by NCI/ADR-RES cells, increasing DOX intracellular accumulation, which supports the greater cell killing ability of these targeted NPs with respect to MDR cells. Altogether, these findings supported the effectiveness of the Tf-surface modification of DOX-PLGA-NPs for an improved antiproliferative activity. Therefore, our pH-responsive Tf-inspired NPs are a promising smart drug delivery system to overcome MDR effect at some extent, enhancing the efficacy of DOX antitumor therapy

PEGylated and poloxamer-modified chitosan nanoparticles incorporating a lysine-based surfactant for pH-triggered doxorubicin release

The growing demand for efficient chemotherapy in many cancers requires novel approaches in target-delivery technologies. Nanomaterials with pH-responsive behavior appear to have potential ability to selectively release the encapsulated molecules by sensing the acidic tumor microenvironment or the low pH found in endosomes. Likewise, polyethylene glycol (PEG)- and poloxamer-modified nanocarriers have been gaining attention regarding their potential to improve the effectiveness of cancer therapy. In this context, DOX-loaded pH-responsive nanoparticles (NPs) modified with PEG or poloxamer were prepared and the effects of these modifiers were evaluated on the overall characteristics of these nanostructures. Chitosan and tripolyphosphate were selected to form NPs by the interaction of oppositely charged compounds. A pH-sensitive lysine-based amphiphile (77KS) was used as a bioactive adjuvant. The strong dependence of 77KS ionization with pH makes this compound an interesting candidate to be used for the design of pH-sensitive devices. The physicochemical characterization of all NPs has been performed, and it was shown that the presence of 77KS clearly promotes a pH-triggered DOX release. Accelerated and continuous release patterns of DOX from CS-NPs under acidic conditions were observed regardless of the presence of PEG or poloxamer. Moreover, photodegradation studies have indicated that the lyophilization of NPs improved DOX stability under UVA radiation. Finally, cytotoxicity experiments have shown the ability of DOX-loaded CS-NPs to kill HeLa tumor cells. Hence, the overall results suggest that these pH-responsive CS-NPs are highly potent delivery systems to target tumor and intracellular environments, rendering them promising DOX carrier systems for cancer therapy

Determination of Methotrexate in pH-Sensitive Chitosan Nanoparticles by Validated RP-LC and UV Spectrophotometric Methods

Nanotechnology-based drug delivery systems are in constant development and, therefore, it is of great importance to have rapid, efficient and accurate analytical methodology to quantify the encapsulated drugs. Here, simple and fast methods, by reversed-phase liquid chromatography (RP-LC) and UV spectrophotometry, were developed and validated for the determination of methotrexate (MTX) in pH-sensitive chitosan nanoparticles (CS-NPs). NPs were prepared using a modified ionotropic complexation process, in which was included a surfactant derived from Nα,Nε-dioctanoyl lysine with an inorganic sodium counterion. The RP-LC method was carried out on a Waters XBridgeTM C18 column (250 mm x 4.6 mm I.D., 5μm), with mobile phase consisted of potassium phosphate buffer (0.05 M, pH 3.2): acetonitrile (86:14, v/v), and UV detection set at 303 nm. The analyses of MTX content by the UV method were also accomplished at 303 nm, using 0.1 M sodium hydroxide as diluent. The measurements were linearly correlated with concentration for both methods in the 1 - 30 μg/mL range (r > 0.9999). The specificity tests showed that there was no interference of the NP components on the quantitative analyses. Precision (repeatability and intermediate precision) was demonstrated by a relative standard deviation lower than 1.5%, whereas the accuracy was assessed by the recovery of MTX from sample matrices, given mean value of ~99%. The proposed methods were applied for the analyses of MTX in different batches of NPs, and the results showed non-significant differences (p > 0.05) between the values obtained with both methodologies. Moreover, the RP-LC method was successfully used to determine the drug entrapment efficiency, and to quantify MTX during in vitro release assays and photolytic degradation studies. In conclusion, the validated methods are suitable to assay MTX in pH-sensitive CS-NPs without any interference from the polymer or surfactant