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Polymeric micelles have found a growing interest as gene vectors due to the serious safety concerns associated with viral vectors. In particular, the cationic polymer polyethylene imine (PEI) has shown relatively high condensation and transfection efficiencies. Additionally, polyethylene glycol (PEG) modification of polymeric gene vectors has dramatically improved their biological properties, including enhanced biocompatibility, prolonged circulation time, and increased bio-distribution. However, PEG grafting of PEI for subsequent condensation of nucleic acids (NAs) does not necessarily result in the formation of a PEI/NAs core with a PEG corona. But often times, the presence of PEG interferes with PEI's electrostatic interaction with NAs. We describe here a facile method to prepare multilayered biodegradable micelles which address some of the critical drawbacks associated with current PEI-based systems. The polyplex micelles have superb stability and stealth properties. Moreover, we describe a method to prepare fully biodegradable and biocompatible injectable hydrogels for use in localized gene therapy.

RNA interference (RNAi) offers a promising base for therapeutic knockdown of clinically relevant genes. Local delivery routes as well as targeted delivery to specific cell populations have been shown to circumvent several hurdles of successful siRNA delivery in vivo. To evaluate and quantify the treatment effect in a precise way, next to measuring the downregulation on gene and protein levels, it is equally essential to investigate the influence on down-stream factors such as generated cytokines. Here, we describe an expressive method to specifically isolate the desired target cells and determine their levels of intracellular cytokines by flow cytometry using the example of murine lungs after pulmonary in vivo transfection with siRNA. Therefore, the lungs of treated mice are harvested and processed into single cell suspensions, in which CD4 positive T cells are marked by antibody-coupled magnetic beads and isolated via magnetic separation. These purified target cells are then fixed and permeabilized, making their intracellular interleukins accessible for staining with fluorescently labeled antibodies. Thus, the cytokine levels and hence the precise influence of the siRNA treatment on intracellular conditions can be measured.