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.

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.

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.

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.

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.