M. Graça Miguel

Cationic agents for DNA compaction

Fluorescence microscopy was used to investigate the conformational changes of individual T4 DNA molecules induced by different compacting agents, namely the cationic surfactants, cetyltrimethylammonium bromide (CTAB) and chloride (CTAC), iron(III), lysozyme, and protamine sulfate. A protocol for establishing size estimates is suggested to obtain reproducible results. Observations show that in the presence of lysozyme and protamine sulfate, DNA molecules exhibit a conformational change from an elongated coil structure to compact globules, usually interpreted as a first-order transition. The maximum degree of compaction that is attained when iron(III) or CTAB (CTAC) are used as compacting agents is considerably smaller, and intermediate structures (less elongated coils) are visible even for high concentrations of these agents. Dynamic light scattering experiments were carried out, for some of the systems, to assess the reliability of size estimates from fluorescence microscopy.

Gel network photodisruption: a new strategy for the codelivery of plasmid DNA and drugs.

In the last 5 years, we have gained further insight on the physical/chemical field of DNA gels. Our expertise on the gel swelling behavior, compaction of DNA by cationic entities, as lipids and surfactants, as well as on the assembly structures of these complexes allow us for the development of novel systems to be used in a variety of biomedical applications. In our previous reports, the physicochemical characterization has been well-established, and now one can evolve to the challenge of using DNA-based carriers in the biological area. Moreover, a new plasmid DNA (pDNA) hydrogel that is porous, is able to swell in the presence of additives, is biocompatible and, thus, is suitable to be used therapeutically was prepared. Here, the dual release of pDNA and solutes with pharmaceutical interest was the main challenge, and thus, we report on the photodisruption of plasmid DNA (pDNA) gels cross-linked with ethylene glycol diglycidyl ether (EGDE) as a strategy for this simultaneous release. The disruption over time, after the irradiation of the gel with ultraviolet light (400 nm), was characterized through the cumulative plasmid DNA release, the evolution in dry weight, the extent of swelling, and also the variations in the gel mesh size. The controlled release of different molecular weight solutes from plasmid DNA gels was investigated, and the influence of both the hydrogel degradation and cross-linker density on the release kinetics were addressed. While the release of lysozyme follows a Fickian process, the release of bovine serum albumin (BSA) and fluoresceinisothiocyanato-dextran (FITC-dextran) is characteristic of a Super Case II release phenomena. In addition, the size of the three solutes partially influences the release behavior; polymer chain mobility and the degree of swelling also play a role. To gain a fundamental understanding of drug release profile from pDNA matrices, in vitro release studies were evaluated using several anti-inflammatory drugs. The quantification of the release mechanism indicates a Super Case II release profile, which can be related with the gel swelling degree. A correlation between the drug release trend and the drug hydrophobicity can be found, with more hydrophobic drugs showing a slower release rate. In brief, this new pDNA gel system is biocompatible, is degradable upon light irradiation, and allows for the controlled and sustained release of plasmid DNA and incorporated solutes. This codelivery of pDNA and drugs would find relevant clinical uses due to the possibility of gene and nongene therapy combination in order to improve the therapeutic efficiency.

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.

DNA gel particles

This review covers recent developments on the topic of DNA gel particles formed in water–water emulsion-type interfaces. A general understanding of interactions between DNA and oppositely charged agents has given us a basis for developing novel DNA-based materials, including gel particles. The association strength, which is tuned 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 DNA (either single- (ssDNA) or double-stranded (dsDNA)). Besides an introduction concerning general aspects about DNA–cationic complexes and the formation of gels in water–water emulsion-type interfaces and some conclusions, the review contains sections reviewing the preparation of DNA gel particles using 1) surfactants, 2) polysaccharides and 3) proteins. The particle morphology, swelling/dissolution behaviour, degree of DNA entrapment and DNA release responses as a function of the nature of the cationic agent used are discussed. Finally, current directions on the preparation of DNA gel particles, including the decrease of size and the improvement of the biocompatibility of these systems have been reviewed.

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.

Plasmid DNA Microgels for a Therapeutical Strategy Combining the Delivery of Genes and Anticancer Drugs

Plasmid DNA (pDNA) is encapsulated into biocompatible microgels by an inverse microemulsion polymerization method. Plasmid DNA and doxorubicin are successfully released from pDNA microgels and their release profiles are characterized by appropriate release models. The co-delivery of genes and drugs from the microgels is evaluated as an enhancer of clinical treatment. Moreover, the release of the encapsulated pDNA is capable of transfection in vitro resulting in the expression of p53 protein. As a whole, a novel pDNA-based system is described that may find biomedical uses, especially in the cancer treatment through the combined action of chemotherapy and gene delivery approach.

Plasmid DNA hydrogels for biomedical applications

In the last few years, our research group has focused on the design and development of plasmid DNA (pDNA) based systems as devices to be used therapeutically in the biomedical field. Biocompatible macro and micro plasmid DNA gels were prepared by a cross-linking reaction. For the first time, the pDNA gels have been investigated with respect to their swelling in aqueous solution containing different additives. Furthermore, we clarified the fundamental and basic aspects of the solute release mechanism from pDNA hydrogels and the significance of this information is enormous as a basic tool for the formulation of pDNA carriers for drug/gene delivery applications. The co-delivery of a specific gene and anticancer drugs, combining chemical and gene therapies in the treatment of cancer was the main challenge of our research. Significant progresses have been made with a new p53 encoding pDNA microgel that is suitable for the loading and release of pDNA and doxorubicin. This represents a strong valuable finding in the strategic development of systems to improve cancer cure through the synergetic effect of chemical and gene therapy.

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.

DNA–poly(vinyl alcohol) gel matrices: Release properties are strongly dependent on electrolytes and cationic surfactants

The release of DNA from cryogel PVA-DNA gel matrices to different electrolyte aqueous solutions was investigated. The rate of release and the distribution coefficient of DNA have been quantified by using a first order kinetic law equation, developed in the frame of a partition-based model. The release of DNA from gels to 1:1 sodium and nitrate salts shows that the transport properties are dependent on the ability of anions/cations to solubilise the DNA in the aqueous phase which, with the exception of bromide, can be related to the Hofmeister series; in the presence of multivalent electrolytes, or increasing the ionic strength, the condensation of DNA inside the gel, followed by a phase separation as seen by scanning electron microscopy, induces the retention of DNA inside the polymer matrix. The DNA condensation and/or phase separation, which contribute to a decrease in the water volume fraction inside the gel, determined by swelling degree experiments, also lead to a decrease in the rate constant of DNA release; such decrease can be justified by the difficulty of the molecular aggregate to move through out the polymeric structure. The DNA release is also dependent on the pH of the bulk solution. The effect of uni- and di-valent cationic surfactants on the release properties of DNA was also evaluated. Our findings suggest that the kinetics of DNA release depends on a complex balance between different structural properties of the surfactants, namely charge, bulkiness of the headgroup and alkyl chain length.

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.