This study proposes a new concept of DNase-triggered delivery systems using polyethylene glycol (PEG)-ssDNA matrices. To construct the matrices, firstly PEG molecules (4 k or 10 k or 35 kDa) were functionalized with single-strand oligonucleotides (PEG-55DNA1). The complete self-assembled matrices were formed by cross-linking of complementary pairing with ssDNA2-conjugated model molecule and subsequent PEG-s5DNA2. The PEG-dsDNA-drug matrices achieved a prolonged release of the tracer molecule (6FAM) in presence of DNase. The release rate of 6FAM exhibited a dependence on the molecular weight of PEG used in matrix formation. A matrix with a higher Mw PEG would provide a slower release of 6FAM due to its denser tangled network being crossed-linked by longer PEG molecules against the approach of DNase. Remarkably, our results showed that nanoscaled complexes (lipoplexes) of lipid-coated PEG-dsDNA-6FAM can be formulated. These lipoplexes no longer release drugs in PBS with DNase but become an efficient vehicle for intracellular drug delivery. We observed a similar prolonged release of 6FAM inside COS-7 cells and noted that the released 6FAM would further distribute to extracellular surroundings. These results indicate that the PEG-ssDNA matrix is able to respond by either external or endogenous DNase activation and becomes an efficient candidate for extracellular or intracellular drug delivery. (C) 2015 Elsevier Ltd. All rights reserved.