Degradable Self-Assembling Dendrons for Gene Delivery: Experimental and Theoretical Insights into the Barriers to Cellular Uptake

Abstract

This paper uses a combined experimental and theoretical approach to gain unique insight into gene delivery. We report the synthesis and investigation of a new family of second-generation dendrons with four triamine surface ligands capable of binding to DNA, degradable aliphatic-ester dendritic scaffolds, and hydrophobic units at their focal points. Dendron self-assembly significantly enhances DNA binding as monitored by a range of experimental methods and confirmed by multiscale modeling. Cellular uptake studies indicate that some of these dendrons are highly effective at transporting DNA into cells (ca. 10 times better than poly(ethyleneimine), PEI). However, levels of transgene expression are relatively low (ca. 10% of PEI). This indicates that these dendrons cannot navigate all of the intracellular barriers to gene delivery. The addition of chloroquine indicates that endosomal escape is not the limiting factor in this case, and it is shown, both experimentally and theoretically, that gene delivery can be correlated with the ability of the dendron assemblies to release DNA. Mass spectrometric assays demonstrate that the dendrons, as intended, do degrade under biologically relevant conditions over a period of hours. Multiscale modeling of degraded dendron structures suggests that complete dendron degradation would be required for DNA release. Importantly, in the presence of the lower pH associated with endosomes, or when bound to DNA, complete degradation of these dendrons becomes ineffective on the transfection time scale-we propose this explains the poor transfection performance of these dendrons. As such, this paper demonstrates that taking this kind of multidisciplinary approach can yield a fundamental insight into the way in which dendrons can navigate barriers to cellular uptake. Lessons learned from this work will inform future dendron design for enhanced gene delivery.

DNA Binding of Pentaamino[60]fullerene Synthesized Using Click Chemistry

“…The critical micelle concentration (CMC) in phosphatebuffered saline, as determined by the solvatochromic shift of the hydrophobic probe Nile red (NR), 18 was 3.6 « 0.5¯M (Figure 1), which is much lower than those of ionic amphiphiles such as SDS (3.5 mM) 19 and CTAB (74¯M), 20 and comparable to high-molecular-weight dendritic amphiphiles. 21 The fluorescence at 638 nm, at concentrations higher than 10¯M, is the same as the value reported for a lipid micelle, 16 which suggests that NR is located in an environment similar to that in a lipid micelle. Comparison of the blue-shift data in Figure 1b with the fluorescence intensity in Figure 1c provides support for this conjecture; that is, below the CMC, the NR fluorescence was partially quenched, 22 which suggested the proximity of NR and the fullerene part of 4, as illustrated on the left of Figure 1c.…”

Nanocarriers for Delivery of siRNA and Co-Delivery of siRNA and Other Therapeutic Agents

“…The system demonstrated low toxicity and high transfection efficiency on human castration-resistant prostate cancer PC-3 cells. Other amphiphilic dendrimers consist of C-18 or cholesterol hydrophobic group and low-generation dendrons have also been synthesized for transfection of siRNA or DNA [122,123]. Although not directly demonstrated in these articles, the hydrophobic core can be used to encapsulate small molecule hydrophobic drugs for siRNA-based co-delivery.…”

Dual-degradable disulfide-containing PEI–Pluronic/DNA polyplexes: transfection efficiency and balancing protection and DNA release

“…[34][35][36] The morphology of multiblock PA-PEI-SS/DNA complexes in aqueous solution was examined by TEM at room temperature. The TEM image showed complex aggregates with a diameter of 150-250 nm ( Figure 5).…”