Tracking the DNA complexation state of pBAE polyplexes in cells with super resolution microscopy

Abstract

The future of gene therapy relies on the development of efficient and safe delivery vectors. Poly(β-amino ester)s are promising cationic polymers capable of condensing oligonucleotides into nanoparticlespolyplexesand deliver them into the cell nucleus, where the gene material would be expressed. The complexation state during the crossing of biological barriers is crucial: polymers should tightly complex DNA before internalization and then release to allow free DNA to reach the nucleus. However, measuring the complexation state in cells is challenging due to the nanometric size of polyplexes and the difficulties to study the two components ( polymer and DNA) independently. Here we propose a method to visualize and quantify the two components of a polyplex inside cells, with nanometre scale resolution, using twocolour direct stochastic reconstruction super-resolution microscopy (dSTORM). With our approach, we tracked the complexation state of pBAE polyplexes from cell binding to DNA release and nuclear entry revealing time evolution and the final fate of DNA and pBAE polymers in mammalian cells. † Electronic supplementary information (ESI) available. See

On Complex Coacervate Core Micelles: Structure-Function Perspectives

“…Whereas oligonucleotides do not need to translocate into the cell nucleus for gene expression, this is essential for DNA. New developments in optical microscopy recently enabled, for the first time, imaging of the fate of internalized C3Ms with nanometric resolution [ 63 , 83 , 84 ]. This shed light on various aspects of nucleus internalization, although much remains to be elucidated.…”

“…pDNA-based C3Ms must remain intact after endosomal escape, because free pDNA is rapidly digested in the cytosol. Two-color direct stochastic optical reconstruction super-resolution microscopy revealed that pDNA-based C3Ms concentrated in the perinuclear region after cell incubation for approximately 12 h [ 84 ]. Passive translocation of DNA-based C3Ms can be achieved in dividing cells, where the nuclear membrane dissociates during cell mitosis.…”

Layer-by-layer pH-sensitive nanoparticles for drug delivery and controlled release with improved therapeutic efficacy in vivo

“…Here, broad-spectrum anticancer drug DOX was used as the model drug, and loaded into the liposome core which was prepared from lipids. The polymer poly(b-amino ester) (PBAE) which has been widely used in drug and/or gene targeted delivery for cancer therapy was selected as pH-responsive layer for drug controlled release (Zhang et al, 2014;Riera et al, 2019). HA, a main component in the extracellular matrix of connective tissue, was selected to form the outside shell of NPs (Lee et al, 2013).…”

Nanoplatforms for mRNA Therapeutics

“…[ 58 ] The polymer chains of PbAE are degradable and can be removed from the cells, leading to low cytotoxicity. [ 59 ] When used in mRNA delivery, basic PbAE exhibits a low efficiency in APCs. [ 60 ] However, PbAE modification is able to enhance the interaction of polyplexes with endosomal membranes and promote endosomal escape.…”