Bioreducible Polymers As a Determining Factor for Polyplex Decomplexation Rate and Transfection

Abstract

Polyplex formation (complexation) and gene release from the polyplexes (decomplexation) are major events in polymeric gene delivery, however the effect of the decomplexation rate on transfection has been rarely investigated. This study employed mixed polymers of poly(L-lysine) (PLL: MW ~7.4 kDa) and reducible PLL (RPLL) (MW ~6.7 kDa) to design decomplexation rate-controllable PLL100-xRPLLx/pDNA complexes (PRLx polyplexes). The transfection efficiency of a model gene (luciferase) in MCF7 and HEK293 cell lines increased with increasing x (RPLL content) in the PRLx polyplexes until peaking at x=2.5 and x=10, respectively, after which point transfection efficiency declined rapidly. In MCF7 cells, PRL2.5 polyplex produced 3- or 223-fold higher gene expression than PLL or RPLL polyplexes, respectively. Similarly, the transfection efficiency of PRL10 polyplex-transfected HEK293 cells was 3.8- or 67-fold higher than that of PLL or RPLL polyplexes, respectively. The transfection results were not apparently related to the particle size, surface charge, complexation/compactness, cellular uptake, or cytotoxicity of the tested polyplexes. However, the decomplexation rate varied by RPLL content in the polyplexes, which in turn influenced the gene transfection. The nuclear localization of pDNA delivered by PRLx polyplexes showed a similar trend to their transfection efficiencies. This study suggests that an optimum decomplexation rate may result in high nuclear localization of pDNA and transfection. Understanding in decomplexation and intracellular localization of pDNA may help develop more effective polyplexes.

Role of Polymeric Endosomolytic Agents in Gene Transfection: A Comparative Study of Poly(l-lysine) Grafted with Monomeric l-Histidine Analogue and Poly(l-histidine)

“…Relative fluorescent units (RFUs) of polyplexes was measured using a plate reader at 495 (excitation) and 515 nm (emission). Free YOYO 1-intercalated pDNA and the buffer solution were set as 100 and 0%, respectively, and pDNA release was calculated by the following equation: 30 …”

“… 29 However, the high positive charge density of PLL still causes cytotoxicity and prevents the release of plasmid DNA (pDNA) from PLL polyplexes. PLL also lacks endosomolytic activity due to the absence of secondary and tertiary amines which results inasmuch as a 10-fold low transfection efficiency in vitro, 30 , 31 than a standard branched poly(ethylenimine) (bPEI 25 K) in polymeric gene transfection. 32 …”

Dexamethasone‐Loaded Reconstitutable Charged Polymeric (PLGA)n‐b‐bPEI Micelles for Enhanced Nuclear Delivery of Gene Therapeutics

“…Cytotoxicity of DexCM was assessed using standard protocols for MTT‐based cell viability assay . MCF7 cells were seeded in 96‐well plates at a density of 5000 cells well −1 in culture medium (0.1 mL).…”

Stimuli-Responsive Gold Nanoparticles for Cancer Diagnosis and Therapy

“…It is the predominant intracellular nonprotein sulfhydryl in animals [ 43 ]. GSH exists in higher concentrations in the cytoplasm and the nucleus (0.5–20 mM) [ 44 , 45 , 46 ] than in in the extracellular fluid (2–20 μM) [ 47 , 48 ]. The importance of GSH lies in its thiol-thiol exchange property, which can break down disulfide bonds and substitute ligands attached on Au surfaces via thiol-Au or other bonds ( Table 3 ).…”