Differential Polymer Structure Tunes Mechanism of Cellular Uptake and Transfection Routes of Poly(β-amino ester) Polyplexes in Human Breast Cancer Cells

Abstract

Successful gene delivery with non-viral particles has several barriers, including cellular uptake, endosomal escape, and nuclear transport. Understanding the mechanisms behind these steps is critical to enhancing the effectiveness of gene delivery. Polyplexes formed with poly(β-amino ester)s (PBAEs) have been shown to effectively transfer DNA to various cell types, but the mechanism of their cellular uptake has not been identified. This is the first study to evaluate the uptake mechanism of PBAE polyplexes and the dependence of cellular uptake on the end group and molecular weight of the polymer. We synthesized three different analogs of PBAEs with the same base polymer poly(1,4-butanediol diacrylate-co-4-amino-1-butanol) (B4S4) but with small changes in the end group or molecular weight. We quantified the uptake and transfection efficiencies of the pDNA polyplexes formulated from these polymers in hard-to-transfect triple negative human breast cancer cells (MDA-MB 231). All polymers formed positively charged (10 – 17 mV) nanoparticles of ~ 200 nm in size. Cellular internalization of all three formulations was inhibited the most (60 – 90% decrease in cellular uptake) by blocking caveolae-mediated endocytosis. Greater inhibition was shown with polymers that had a 1-(3-aminopropyl)-4-methylpiperazine end group (E7) than the others with a 2-(3-aminopropylamino)-ethanol end group (E6) or higher molecular weight. However, caveolae-mediated endocytosis was generally not as efficient as clathrin-mediated endocytosis in leading to transfection. These findings indicate that PBAE polyplexes can be used to transfect triple negative human breast cancer cells, and that small changes to the same base polymer can modulate their cellular uptake and transfection routes.

Polymeric nanoparticles as cancer-specific DNA delivery vectors to human hepatocellular carcinoma

“…Corroborative findings of Kim et al [39] reveal that the specific cellular uptake pathways employed by PBAE NPs can also be critical. In this work, although the preferential uptake mechanism for PBAE NPs in human triple-negative breast cancer cells was observed to be caveolae-mediated, uptake through the clathrin pathway was suggested to be more efficient, leading to relatively higher transfection levels [39].…”

“…Corroborative findings of Kim et al [39] reveal that the specific cellular uptake pathways employed by PBAE NPs can also be critical. In this work, although the preferential uptake mechanism for PBAE NPs in human triple-negative breast cancer cells was observed to be caveolae-mediated, uptake through the clathrin pathway was suggested to be more efficient, leading to relatively higher transfection levels [39]. Sunshine et al [38] and Bhise et al [40] suggest that the end-capping of PBAEs is a critical parameter for increasing the cellular uptake of PBAE NPs as well and this is an intriguing area of research for future studies.…”

Complexation and release of DNA in polyplexes formed with reducible linear poly(β-amino esters)

“…To improve the solubility and to avoid such aggregates, we acidified the pH two units (to pH 5), making the amine groups in the polymer more charged and, subsequently, making the whole molecule more polar. Several studies on poly(-amino esters) have previously encountered this solubility issue and the resulting Page 10 of 33 A c c e p t e d M a n u s c r i p t 10 polyplexes have been formulated in acetate buffer at pH 5 [12,40,41]. The pH change (from 7 to 5) immediately translated into an almost complete solubilization of the polycations (see Fig.…”

A Triple-Fluorophore-Labeled Nucleic Acid pH Nanosensor to Investigate Non-viral Gene Delivery

“…42 A related aspect is that the type of endocytosis, such as clathrin-mediated versus caveolae-mediated, can also make a significant difference to the efficiency of transfection, and that this can occur in a polymer structure-specific manner. 43 Nonetheless, it is clear that sequestration of delivered DNA into acidic late endosomes and lysosomes means that those DNA molecules are not available to be transcribed in the pH-neutral nucleus, the target of the gene delivery. Thus, this nanobiosensor can play an important role in probing this barrier in a high-throughput, single-cell, and dynamic fashion.…”