Abstract
Polyplexes are nanoparticles formed by the self-assembly of DNA/RNA and cationic polymers specifically designed to deliver exogenous genetic material to cells by a process called transfection. There is a general consensus that a subtle balance between sufficient extracellular protection and intracellular release of nucleic acids is a key factor for successful gene delivery. Therefore, there is a strong need to develop suitable tools and techniques for enabling the monitoring of the stability of polyplexes in the biological environment they face during transfection. In this work we propose time-resolved fluorescence spectroscopy in combination with SYBR Green I-DNA dye as a reliable tool for the in-depth characterization of the DNA/vector complexation state. As a proof of concept, we provide essential information on the assembly and disassembly of complexes formed between DNA and each of three cationic polymers, namely a novel promising chitosan-graft-branched polyethylenimine copolymer (Chi-g-bPEI), one of its building block 2 kDa bPEI and the gold standard transfectant 25 kDa bPEI. Our results highlight the higher information content provided by the time-resolved studies of SYBR Green I/DNA, as compared to conventional steady state measurements of ethidium bromide/DNA that enabled us to draw relationships among fluorescence lifetime, polyplex structural changes and transfection efficiency.
Highlights
The ease of synthesis, relatively low cost and high safety make cationic polymers attractive and valuable tools for the delivery of nucleic acids to cells,[2] their widespread use in clinical trials has mainly been hampered by transfection efficiency concerns
Polyethylenimine (PEI) is considered as the gold standard gene carrier among polymeric non-viral vectors. It exists in either linear or branched form and, conflicting results are reported in the literature, several authors have pointed out the superior transfection efficiency of bPEI.[5]
Both the transfection efficiency and cytotoxicity of PEIs are strictly related to their molecular weight (Mw): high Mw (HMW) PEIs lead to enhanced transfection and induce undesirable cytotoxicity.[6,7]
Summary
The ease of synthesis, relatively low cost and high safety make cationic polymers attractive and valuable tools for the delivery of nucleic acids to cells,[2] their widespread use in clinical trials has mainly been hampered by transfection efficiency concerns These gene delivery vectors are inherently cationic at physiological pH, spontaneously self-organizing with polyanionic nucleic acids in nano- and microparticles named polyplexes.[3,4] In this scenario, there is a growing consensus that a better understanding of the processes leading to DNA complexation/release and to effective polyfection represents a big step forward in the rational design of effective polymeric gene vectors. Polyethylenimine (PEI) is considered as the gold standard gene carrier among polymeric non-viral vectors It exists in either linear (lPEI) or branched (bPEI) form and, conflicting results are reported in the literature, several authors have pointed out the superior transfection efficiency of bPEI.[5]. Both the transfection efficiency and cytotoxicity of PEIs are strictly related to their molecular weight (Mw): high Mw (HMW) PEIs lead to enhanced transfection and induce undesirable cytotoxicity.[6,7] Recently, aiming to reduce the toxic
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