Andreia F. Jorge

DNA condensation by pH-responsive polycations.

This work addresses the impact of pH variation on DNA-polyethylenimine (PEI) complex formation, in aqueous solution and at constant ionic strength. An initial potentiometric characterization of the acid-base behavior of PEI is carried out to measure the concentration of ionized species in the relevant systems. The characterization of the DNA-PEI complexes is performed by precipitation assays, agarose gel electrophoresis, photon correlation spectroscopy, and zeta potential analysis. It is observed that the variations on the electrophoretic mobility, size, and electrical properties of complexes display nonmonotonic, nontrivial trends with pH, if the same polycation/polyanion charge ratios are used for different values of pH. It is seen that both linear charge density and the relative number of chains of the condensing agent are important factors governing the condensation behavior. Complexes prepared at pH 4, for example, indicate strong binding and a large mean size, while those prepared at pH 8 are smaller, in a more uniform population. Finally, charge inversion was observed for all studied pH values (even below charge neutralization).

Characterization of polyplexes involving small RNA

The purpose of the present study is to provide a tool for an efficient design and synthesis of non-viral vectors for small RNA delivery. The effects of properties of the polycation, such as molecular weight, charge density and backbone structure, to polyplex structure and physicochemical behavior were systematically evaluated. The condensing agents, polyethylenimine (PEI), chitosan (CS) and poly(allylamine) (PAA) were added to sRNA molecules at different N/P ratio. The efficiency of encapsulation and protection of sRNA, as well as polyplex size, zeta potential and morphology were followed and compared. The results show that PEI/sRNA polyplexes display a small size and positive zeta potential. However, for low molecular weights, this polycation is unable to protect sRNA in the presence of a decompacting agent. With chitosan, sRNA is efficiently compacted at high N/P ratios. The CS/sRNA complexes display small sizes, ca. 200 nm, positive surface charge and also good stability. Finally, the PAA/sRNA polyplexes were found to be the smallest at low N/P ratios, displaying a good encapsulation efficiency and high stability. A rationale for the experimental observations is provided using Monte Carlo simulation for systems with polycations of different length and charge density. The simulations showed that there is an interplay between the size of polycation chains and its charge density that define the degree of condensation for sRNA.

Enhanced condensation and facilitated release of DNA using mixed cationic agents: a combined experimental and Monte Carlo study.

Efficient DNA condensation and decondensation, as well as low toxicity, are required for an efficient gene delivery vehicle. We report on the condensation of DNA by a mixture of cationic agents, low-molecular-weight polyethylenimine (PEI, 1.2 KDa) and Fe(III) ions, and respective decondensation, using experimental and theoretical methods. It was found that a significant reduction in the amount of PEI necessary to induce DNA condensation is achieved by the addition of the trivalent ions, which are very inefficient on their own. In addition, the mixture makes DNA decompaction by heparin easier, starting from similar degrees of condensation. The results obtained using simulations of coarse-grain models are coherent with those obtained experimentally. It was also found that the improved effect of the multivalent ions is related to the preferred positioning of the trivalent ions in the DNA areas less populated by the polycation chains, in between the polycation chains and at the ends of the DNA, which facilitates the overall condensation.

Interpreting the Rich Behavior of Ternary DNA-PEI-Fe(III) Complexes

This work aims to shed light on the mechanism of interaction between components of ternary DNA-PEI-Fe(III) complexes, using experimental and theoretical approaches. In the experimental part, the chelation between PEI-Fe(III) was inspected by potentiometry and electrical conductance measurements and the respective importance for the condensation of DNA analyzed. To this end, three different mixing protocols for the components were imposed using different PEIs, branched (bPEI1.2 and bPEI10) and linear (lPEI2.5 and lPEI25). A delay in DNA condensation was observed when PEI and Fe(III) were premixed and then added to DNA. The set of observations was complemented by determination of the amount of Fe(III) included in the polyplexes, which was found to be dependent on the order of mixture and on the type of PEI used, decreasing with intrinsic PEI condensation efficiency. Overall, a coherent picture in which Fe(III) compensates PEI, probably modulating the respective charge, emerges. Some points arisen from the experimental part were rationalized using Monte Carlo simulations. Different architectured polycation (PC) chains were modeled and an interaction between PC and multivalent ions, mimicking the chelation of Fe(III) by the PEI, was imposed. It was found that chelation enhances polyanion (PA) compaction, irrespective of the PC architecture and charge density. The amount of multivalent ions in each polyplex compensates the negative charge unbalanced by the PC. The charge density and the ability of chelation of each PC dictate the disposition of each condensing agent along the PA backbone, and their coexistence strengthens PA compaction. The deep understanding of these ternary mixtures is a step forward in the optimization of such systems for application in gene delivery.

Covalent Strategies for Targeting Messenger and Non-Coding RNAs: An Updated Review on siRNA, miRNA and antimiR Conjugates

Oligonucleotide-based therapy has become an alternative to classical approaches in the search of novel therapeutics involving gene-related diseases. Several mechanisms have been described in which demonstrate the pivotal role of oligonucleotide for modulating gene expression. Antisense oligonucleotides (ASOs) and more recently siRNAs and miRNAs have made important contributions either in reducing aberrant protein levels by sequence-specific targeting messenger RNAs (mRNAs) or restoring the anomalous levels of non-coding RNAs (ncRNAs) that are involved in a good number of diseases including cancer. In addition to formulation approaches which have contributed to accelerate the presence of ASOs, siRNAs and miRNAs in clinical trials; the covalent linkage between non-viral vectors and nucleic acids has also added value and opened new perspectives to the development of promising nucleic acid-based therapeutics. This review article is mainly focused on the strategies carried out for covalently modifying siRNA and miRNA molecules. Examples involving cell-penetrating peptides (CPPs), carbohydrates, polymers, lipids and aptamers are discussed for the synthesis of siRNA conjugates whereas in the case of miRNA-based drugs, this review article makes special emphasis in using antagomiRs, locked nucleic acids (LNAs), peptide nucleic acids (PNAs) as well as nanoparticles. The biomedical applications of siRNA and miRNA conjugates are also discussed.

Sustainable DNA Release from Chitosan/Protein Based-DNA Gel Particles

Chitosan lactate (CL) alone and in combination with protamine sulfate (PS) was used as an intrinsic biocompatible carrier to form DNA gel particles by interfacial diffusion. Protamine sulfate is highly positively charged, arginine-rich protein, which has been previosly used in the formation of mixed carriers for modulating DNA release. In view of the promising properties of oligosaccharides and the well-known cell-penetrating and nuclear localization capabilities of protamines, we presume that both structures could play a critical role in DNA delivery. The purpose of this study was to evaluate the capability of water-soluble, low molecular weight chitosan lactate to form DNA gel particles alone (binary system) and in combination with the protein protamine sulfate (ternary system). The particles were characterized with respect to the degree of DNA entrapment, the swelling and dissolution behavior, the secondary structure of DNA in the particles, and the kinetics and mechanisms of DNA release. We controlled the magnitude of DNA release and achieved controlled release by using mixed systems and changing the CL/PS ratio in the solution where the particles were formed. The Rose Bengal partition assay was applied for the first time to estimate the surface hydrophobicity of DNA gel particles. Both CL alone and in combination with PS promotes the formation of DNA gel particles that have an acute hydrophilic character, which may govern the posterior adsorption of plasma proteins and influence the bioavailability of the systems. The lack of hemolytic effect of these DNA gel particles suggests their potential application as long-term blood-contacting medical devices.

Ternary complexes DNA–polyethylenimine–Fe(III) with linear and branched polycations: implications on condensation, size, charge and in vitro biocompatibility

The successful use of nucleic acids in medicine requires efficient DNA condensation and protection, as well as a low cytotoxicity of the vector. In previous work, we have used a ternary DNA–PEI–Fe(III) complex and have shown that the addition of Fe(III) to the DNA–PEI complexes enhances DNA condensation, whereas allows an easier DNA release from the complexes, in the presence of a decompacting agent. The focus of the present work was to investigate the influence of the polycation architecture on the DNA–PEI–Fe(III) ternary system, and also to develop a potential gene delivery system with reduced toxicity. Herein, lPEI (2.5 and 25 kDa) and bPEI (1.2 and 10 kDa) were used in conjunction with Fe(III), and the complexes formed were physico-chemically characterized using precipitation and gel electrophoresis assays, and size and zeta potential determinations. It was observed that the addition of Fe(III) ions equalizes the charge ratios for the onset of DNA condensation irrespective of the PEI used, although a more pronounced condensation effect is still visible with the polycations with a lower Mw. At low N/P ratios, the presence of Fe(III) caused the zeta potential to change from negative to positive values. Also, a reduction in polyplex size is visible before aggregation takes place. The hemolytic activity of the condensing agents was found to be dependent on the pH of the medium, but no perturbation of erythrocytes integrity was detected. Furthermore, the cytotoxicity of each condensing agent individually and of the complexes towards 3T3 fibroblast cell line and HeLa cell line was monitored using the MTT and LDH assays. The magnitude of the cytotoxic effects of the polycations and Fe(III) was found to be dependent on the concentration of the condensing agents, the molecular weight of the polycations, and the pH of the culture medium. The combination of Fe(III) with PEI determines higher IC50 values than in the absence of the metal ion. Additionally, a lower cytotoxicity of DNA–PEI–Fe(III) complexes was observed, when compared to native PEI–DNA complexes, at the same degree of condensation and values of zeta potential. The results show that Fe(III) reinforces the DNA–PEI condensation in both PEI architectures. It allows the cytotoxicity of PEIs to be mitigated and the concentration of the latter needed to obtain stable polyplexes to be decreased.

Pollen flow between flowers of the same morph in invasive populations of Oxalis pes-caprae L. in the western Mediterranean region

Oxalis pes-caprae is a tristylous South African geophyte that is invasive in regions with Mediterranean climate. Given the introduction of the short-styled morph only, vegetative reproduction was, until recently, the reproductive mode known for this invaded area. The detection of new floral morphs, fruit and seed production in natural populations of the invaded range, together with the reported weakening in the heteromorphic incompatibility system, raised the question on whether the short-styled flowers are able to exchange pollen among themselves, and if so, which mechanisms account for it. Thus, flower morphometric analysis and field experiments were conducted to assess pollinator visitation rates and pollen flow in three invasive populations. Flower morphological changes suggesting the evolution from tristyly towards semi-homostyly were observed. Moreover, O. pes-caprae flowers were visited by different functional groups of pollinators, mostly hymenopteran. Dye flow experiments revealed that the short-styled flowers were able to successfully exchange pollen among themselves in natural conditions. All of this, together with the weakening of the incompatibility system, constitutes further data that might help to explain the recently reported occurrence of sexual reproduction in this invaded range, which by itself bears important implications for the invasion process of O. pes-caprae.

Flower biology and breeding system of Salvia sclareoides Brot. (Lamiaceae)

A remarkable diversity of floral adaptations and reproductive strategies has evolved in flowering plants as a result of natural selection mediated by pollinators. The form and functioning of sexual units determine the patterns of pollen dispersal and receipt and, together with post-pollination mechanisms including incompatibility systems, determine the plant reproductive fitness. Reproductive biology assumes a special importance in rare and endemic species due to restricted population/distribution sizes. Salvia sclareoides Brot. (Lamiaceae) is an endemic species of Iberian Peninsula with conservation interest for which little information is available. The aim of this work was to study the flower biology and breeding system of S. sclareoides and assess how reproductive traits, pollination services and herbivory affected the reproductive success of this species. One natural population was selected to study flower lifespan, floral rewards and sexual functioning and to determine the reproductive system; additionally, in three natural populations, pollen limitation and herbivory of sexual structures were quantified. Salvia sclareoides presents short-lived, nectar rewarding flowers arranged in inflorescences with several flowers simultaneously open. The flowers are protandrous, although the peak of pollen germinability and stigmatic receptivity coincided. Pollination experiments revealed that S. sclareoides is self-compatible, having some ability of spontaneous selfing. Still, the presence of pollinators significantly increased the reproductive success. Also, both quantitative and qualitative pollen limitation was observed. Finally, it was revealed that the reproductive success was also affected by herbivores that damaged significantly the sexual structures. The consequences of the obtained results for the dynamics of S. sclareoides populations are discussed.

Combining polyethylenimine and Fe(III) for mediating pDNA transfection

BACKGROUND The potential use of Fe(III) ions in biomedical applications may predict the interest of its combination with pDNA-PEI polyplexes. The present work aims at assessing the impact of this metal on pDNA complex properties. METHODS Variations in the formation of complexes were imposed by using two types of biological buffers at different salt conditions. The incorporation of pDNA in complexes was characterised by gel electrophoresis and dynamic light scattering. Transfection efficiency and cytotoxicity were evaluated in HeLa and HUH-7 cell lines, supported by flow cytometry assays. RESULTS Fe(III) enhances pDNA incorporation in the complex, irrespective of the buffer used. Transfection studies reveal that the addition of Fe(III) to complexes at low ionic strength reduces gene transfection, while those prepared under high salt content do not affect or, in a specific case, increase gene transfection up to 5 times. This increase may be a consequence of a favoured interaction of polyplexes with cell membrane and uptake. At low salt conditions, results attained with chloroquine indicate that the metal may inhibit polyplex endosomal escape. A reduction on the amount of PEI (N/P 5) formed at intermediary ionic strength, complemented by Fe(III), reduces the size of complexes while maintaining a transfection efficiency similar to that obtained to N/P 6. CONCLUSIONS Fe(III) emerges as a good supporting condensing agent to modulate pDNA-PEI properties, including condensation, size and cytotoxicity, without a large penalty on gene transfection. GENERAL SIGNIFICANCE This study highlights important aspects that govern pDNA transfection and elucidates the benefits of incorporating the versatile Fe(III) in a gene delivery system.