Transfection activity and the mechanism of pDNA-complexes based on the hybrid of low-generation PAMAM and branched PEI-1.8k

Abstract

Cationic polymers have been regarded as promising non-viral gene carriers because of their advantages over viral gene vectors, such as low cost, a high level of safety and easy manipulation. However, their poor transfection efficiency in the presence of serum and high toxicity are still limiting issues for clinical applications. In addition, the lack of adequate understanding of the gene delivery mechanism hinders their development to some extent. In this study, new polycations (PAPEs) consisting of a low generation polyamidoamine (PAMAM) core and branched polyethyleneimine (PEI-1.8k) outer layers were synthesized and their transfection activity and mechanism were studied. PAPEs were characterized by FTIR, (1)H NMR and gel permeation chromatography. PAPEs were able to self-assemble with pDNA and form spherical nanoparticles with sizes of 70-204 nm and zeta potentials of 13-33 mV. Importantly, the PAPE-pDNA complexes displayed lower cytotoxicity and higher transfection activity than PEI 25k in various cell lines, specifically in the presence of serum. The transfection mechanism was evaluated by endocytosis inhibition with specific inhibitors, time-dependent transfection, and intracellular trafficking inspection by CLSM. The high levels of transgene expression mediated by PAPEs were attributed to caveolae-mediated cellular uptake, the reduced entry into lysosomes and the entry into the nucleus through mitosis.

Spectroscopic and calorimetric studies of formation of the supramolecular complexes of PAMAM G5-NH2 and G5-OH dendrimers with 5-fluorouracil in aqueous solution

“…S1) as well as the mixtures of this drug and PAMAM G5-OH (Fig. S2) in heavy water as in previous 1 H NMR studies of PAMAM-NH 2 (Bányai et al, 2013;Barra et al, 2014a,b;Buczkowski et al, 2012bBuczkowski et al, , 2013Cao et al, 2013;Giri et al, 2011;Hu et al, 2010Hu et al, , 2012Keri et al, 2015) and PAMAM-OH dendrimers (Choi et al, 2012;Giri et al, 2011;Gomez et al, 2009a,b;Hu et al, 2012;Pellechia et al, 2004;Wang et al, 2013). With increasing the concentration of 5-fluorouracil in the mixture of drug and dendrimer one can observe that peak at 2.6 ppm corresponding to the protons of methylene groups present at tertiary amine groups is not clearly shifted during the drug bonding by cationic PAMAM G5-NH 2 dendrimer (Fig.…”

“…Literature presents numerous reports on possible methods of using these macromolecules (Bhattacharya et al, 2013), among other things in biology or medicine Gor et al, 2014;Liu et al, 2014;Zhao et al, 2011). Dendrimer macromolecules can fulfil the function of supramolecular nanocarriers of low-molecular ligands: fragments of genetic material (Cao et al, 2013;Eichman et al, 2000;Jang et al, 2009;Pavan et al, 2010a,b;Peng et al, 2010;Shakhbazau et al, 2010;Wang et al, 2010), medical image contrasts (Tomalia et al, 2007) or drugs (Cheng and Xu, 2005;D'Emanuele and Attwood, 2005;Gupta et al, 2006;Kesharwani et al, 2014;Medina and El-Sayed, 2009;Najlah and D'Emanuele, 2006), including oncological drugs Kong et al, 2014;Sadekar et al, 2013;Thomas et al, 2010). Dendrimers can also influence the conformation of protein being of importance for the functioning of protein nervous system (Klajnert et al, 2007;Neelov et al, 2013), reduce the risk of viral infections (Tyssen et al, 2010), including HIV (Date and Destache, 2013;Tyssen et al, 2010;Vacas-Córdoba et al, 2014) and facilitate the permeation of substances through cell membranes (Ainalem et al, 2010;Kelly et al, 2008;Larson, 2006, 2008a,b;Mecke et al, 2004;Tiriveedhi et al, 2011;Voulgarakis et al, 2009) and skin Perumal, 2008, 2009;Yang et al, 2012).…”

Divalent Folate Modification on PEG: An Effective Strategy for Improving the Cellular Uptake and Targetability of PEGylated Polyamidoamine–Polyethylenimine Copolymer

“…At the N/P ratio of 10, the mean sizes decreased to below 200 nm, which is very suitable for cellular uptake and transport through the cytoplasm. 42 More importantly, all complexes had low polydispersity index (PDI) Figure 6. Comparison of cellular uptake of DNA complexes with PME−(PEG 3.5k ) 1.69 , PME−(PEG 3.4k -FA 1 ) 1.66 , and PME−(PEG 3.4k -FA 2 ) 1.72 in HeLa cells.…”

An efficient method for in vitro gene delivery via regulation of cellular endocytosis pathway

“…[29][30][31] Endocytosis inhibitors were usually applied to analyze the endocytosis pathways of cells, but their concentrations and treatment time should be well determined to minimize cytotoxicity. 32,33 Meanwhile, inhibition of certain endocytosis pathways may artificially upregulate other internalization routes that are not originally involved in the uptake of transfection particles because many chemical inhibitors are not specific to a single internalization pathway. 27 Therefore, inhibition experiments should be rationally designed to observe effects at a suitable time in referable parallel treatments.…”