Diana Costa

Swelling behavior of a new biocompatible plasmid DNA hydrogel

Chemical plasmid DNA (pDNA) gels were prepared by a cross-linking reaction with ethylene glycol diglycidyl ether (EGDE). Fluorescence microscopy (FM) and scanning electron microscopy (SEM) images of pDNA gels are reported. For the first time, the pDNA gels have been investigated with respect to their swelling in aqueous solution containing different additives, such as metal ions, polyamines, polycations and surfactants. The effect of the cationic surfactant tail length on the volume phase transition of pDNA gels was studied as a function of surfactant concentration; the critical aggregation concentration (cac), is found to decrease with increasing length of the hydrophobic tail. The deswelling appears to be reversible as exemplified by the addition of anionic surfactant subsequent to collapsing the gel by a cationic surfactant. Cell viability assays suggest that the plasmid DNA gels are non-toxic to cells and do not cause any distinct harm to them. This step contributes to the possibility of using these gels, as carriers, in real biological systems.

Swelling properties of cross-linked DNA gels.

This work represents our contribution to the field of physical chemistry of DNA gels, and concerns the synthesis and study of novel chemically cross-linked DNA gels. The use of covalent DNA gels is a very promising way to study DNA-cosolute interactions, as well as the dynamic behaviour of DNA and cationic compacting agents, like lipids, surfactants and polycations. Manipulating DNA in new ways, like DNA networks, allows a better understanding and characterization of DNA-cosolute complexes at the molecular level, and also allows us to follow the assembly structures of these complexes. The use of responsive polymer gels for targeted delivery of toxic and/or labile drugs has, during the past few years, shown to be a promising concept. The features found in the proposed system would find applications in a broader field of gel/drug interaction, for the development of controlled release and targeted delivery devices.

Stimuli-responsive polyamine-DNA blend nanogels for co-delivery in cancer therapy

Polyamine plasmid DNA (pDNA) hydrogels have been synthesized by an original approach which conjugates pDNA condensation by polyamines and cross-linking reaction with ethylene glycol diglycidyl ether. In an attempt to design more sophisticated vectors with enhanced transfection efficiency and targeting ability, the cell-binding ligand transferrin has been incorporated into polyethylenimine formulations. All systems are photodegradable which allows for the controlled release of different plasmids (pVAX1-LacZ and pcDNA3-FLAG-p53) and anticancer drugs (doxorubicin, epirubicin and paclitaxel). The tumoral treatment through the combined action of pcDNA3-FLAG-p53 gene and an anticancer drug has a stronger potential to suppress the development of cancer cells. The effect is greatly improved when transferrin is encapsulated into the carriers. This study is a relevant contribution for the design of novel generation of plasmid biopharmaceuticals for progresses in gene cancer therapy, feeding the hope of cancer cure.

Rhodamine based plasmid DNA nanoparticles for mitochondrial gene therapy

Conventional treatments for patients suffering from mitochondrial cytopathies are, in most of the cases, inefficient and there is, until now, no effective cure. Mitochondrial gene therapy can be seen as a valuable approach to reestablish normal metabolic function, adding a new perspective of treatment for mitochondrial-related diseases. We developed novel mitochondrial-targeted plasmid DNA nanoparticles by incorporation of rhodamine 123, a fluorescent amphiphile with mitochondria affinity. These nanocarriers have suitable sizes for gene therapy purposes, are biocompatible and are able to protect the encapsulated pDNA from nucleases digestion. Furthermore, the pDNA vectors were easily internalized intodifferent cell linesand targeted delivery to mitochondria was confirmed by fluorescence confocal microscopy. In addition, p53 protein inexpression, mediated by rhodamine nanoparticles, demonstrates the ability of the proposed system to target mitochondria; due to the different genetic code in mitochondria, p53 protein cannot be expressed. Overall, the presented model pDNA constructs possess interesting properties as gene delivery systems and their mitochondrial target ability might have a profound relevance for further engineering of adequate vectors to be applied in mitochondrial gene therapy field.

Polyethylenimine coated plasmid DNA–surfactant complexes as potential gene delivery systems

Nanometer scaled particles have been prepared from strong association between plasmid DNA (pcDNA3-FLAG-p53) and oppositely charged surfactants. Although these particles present suitable properties for gene delivery purposes, their cytotoxicity could compromise their use in gene therapy applications. To ensure biocompatibility of this potential gene delivery system, the nanoparticles were coated with polyethylenimine (PEI) with various molar ratios of PEI nitrogen to plasmid DNA phosphate groups. This led to a drastic increase in the cell viability of the particles, and in addition particle characteristics such as size, surface charge and loading efficiency, have also been enhanced as a result of the PEI coating process. The dissolution or swelling/deswelling behaviour displayed by these particulate vehicles could be tailored and monitored in time, to promote the controlled and sustained release of plasmid DNA. Moreover, we show that both the surfactant alkyl chain length and the ratio of nitrogen to phosphate groups are important parameters for controlling the plasmid DNA release. Overall, the developed plasmid DNA carriers have the potential as a new nanoplatform to be further explored for advances in the gene therapy field.

Plasmid DNA nanogels as photoresponsive materials for multifunctional bio-applications.

This study provides a detailed description on the synthesis and characterization of novel polyamine plasmid DNA nanogels. Ethylene glycol diglycidyl ether was used as cross-linker, in conjugation with polyamines to promote pDNA condensation. The biocompatible nanovectors exhibit a unique swelling behavior in water and salt solutions. These systems are light photodegradable allowing their use in a broad range of biotechnological applications. Different plasmids, pVAX1-LacZ and pcDNA3-FLAG-p53, and anticancer drugs were, thus, efficiently loaded in the nanogels and their controlled release was demonstrated. Furthermore, the dual delivery of pcDNA3-FLAG-p53 gene and anticancer drugs illustrates the possibility of the combination of chemical and gene therapies. This new versatile and easy method of nanohydrogels preparation provides a potential synthetic approach for the design of tunable systems which can display multiple functions, sensitivity to different stimuli and exhibit programmed responses as well.

Modeling the Surfactant Uptake in Cross-Linked DNA Gels

The deswelling behavior of cross-linked DNA gels reports on DNA-cosolute interactions and gives a basis for the development of responsive DNA formulations. An investigation of the deswelling kinetics shows that an increase in the surfactant tail length gives a pronouncedly slower deswelling kinetics. In the same conditions, single stranded gels exhibited faster deswelling kinetics when compared with double stranded networks. It was also found that DNA gels display a nonmonotonic volume change with time, deswelling followed by reswelling, when immersed in surfactant solutions. Kinetic modeling of surfactant uptake by the DNA gel was done using a stochastic approach of mass transfer between the bulk solution, the surface, and the inner volume of the gels. Diffusion coefficients and kinetic constants can be derived from the separated model uptake curves for the gel surface and the gel interior volume.

DNA-Based Hydrogels: An Approach for Multifunctional Bioapplications

DNA-based networks have attracted significant interest in the last decades, due to its hydrophilicity, biocompatibility and stimuli responsiveness. These characteristics make them very suitable for a variety of applications in the biomedical field. In this context, relevant advances on the design and formulation of DNA-based systems as technological devices to be used in clinical applications have been accomplished. In the last few years, particular attention has been focused on the plasmid DNA (pDNA) hydrogels. Biocompatible pDNA gel networks were synthesized by a cross-linking reaction. In order to enhance transfection efficiency and targeting of the systems, transferrin has been included in the protocol of hydrogels preparation. All developed carriers are photodegradable which opens the possibility for the sustained and controlled delivery of different plasmids and anticancer drugs. The cancer treatment approach based on the combination of chemotherapy and specific gene delivery demonstrated to possess stronger ability to weaken the growth and proliferation of tumour cells. The effect is enhanced when transferrin is present in the pDNA hydrogels. This finding is a great achievement and instigates further research focused on the generation of new vectors for the delivery of biopharmaceuticals contributing for the evolution of cancer therapy.