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The main objective of the present work was the development of new nanoparticulate carrier systems for the delivery of plasmid DNA. These new carriers consist of a blend matrix formed by a poly(lactic-co-glycolic acid) (PLGA) copolymer and polyoxyethylene derivatives. More specifically, we have prepared nanostructures with different PLGA:poloxamer and PLGA:poloxamine compositions by an optimized emulsification-solvent diffusion technique and studied the potential of these carriers for the encapsulation and controlled release of plasmid DNA. Depending on the particle composition, the encapsulation efficiency of the model plasmid pEGFP-C1 varied between 30% and 45%. All formulations provided continuous and controlled release of the plasmid with minimal burst effect. In addition, the release rate and duration was dependent on the composition of the particle matrix. Moreover, gel electrophoresis and cell culture (MCF-7 cell line) assays allowed us to confirm that the biologically active form of the plasmid was preserved during the particle preparation process and also during its release. Cell culture experiments also indicated that the new nanoparticles do not exhibit toxic effects on these cells at concentrations up to 5 mg/mL. Altogether, these results indicate that these composite nanostructures present a promising approach for the delivery of plasmid DNA.