Insights in cellular uptake mechanisms of pDNA–polycationic amphiphilic cyclodextrin nanoparticles (CDplexes)

Abstract

have equally contributed to the reported work. Abstr actIt is generally recognized that the major obstacle to efficient gene delivery is cellular internalization and endosomal escape of the DNA. Recently, we have developed a modular strategy for the preparation of well-defined polycationic amphiphilic cyclodextrins (paCDs) capable of complexing and compacting DNA into homogeneous nanoparticles (<70 nm).Since paCDs resemble both cationic polymers and cationic lipids, it is conceivable that the corresponding pDNA-paCD nanoparticles (CDplexes) might use the cell uptake and endosomal escape mechanisms described for both lipoplexes and polyplexes. To verify this hypothesis, we have now investigated the uptake and transfection efficiencies of CDplexes in the presence of several inhibitors of endocytosis, namely chlorpromazine, genistein, dynasore and methylated β-cyclodextrin (MbCD). Our data show that CDplexes obtained from paCD 1, which ranks among the most efficient paCD gene vectors reported up to date, are internalized by both clathrin-dependent (CDE) and clathrin-independent endocytosis (CIE), both processes being cholesterol-and dynamin-dependent. We observed that the largest fraction of gene complexes is taken up via CDE, but this fraction is less relevant for transfection. The smaller fraction that is internalized via the CIE pathway is predominantly responsible for successful transfection. Intr oductionThe successful delivery of therapeutic genes into cells and their availability at the intracellular site of action are crucial requirements for successful gene therapy. During the last decade, under the advent of nanotechnology, a broad diversity of creative materials featuring promising properties for nonviral gene delivery applications has emerged, (1). Cationic polymers and lipids, or their combinations, have shown the ability to bind nucleic acids through electrostatic interactions and condense them into complexes that can be readily internalized by cells (2-3). The gene delivering capabilities of many of these systems have been thoroughly investigated. Yet, drawing firm conclusions that might serve to provide feedback for the design of improved delivery entities is a delicate issue as a consequence of the essentially polydisperse nature of such structures (4).The control of the architecture of multifunctional macromolecules is a major determinant in the rational design of successful nonviral gene delivery systems. Novel 3 strategies have been implemented to build up well defined constructs featuring selfassembling capabilities in the presence of nucleic acids. In this context, the unique characteristics of dendrimers and other highly ordered clusters, such as uniformity, monodispersity and multivalency, have attracted increasing attention (5-8). Homogeneous external functionalization of these platforms becomes, however, exponentially more complicated as the dendrimer generation increases (9-10). The use of preorganized macrocyclic scaffolds, such as calixarenes, to achieve a precise alignment of functiona...

Mechanistic studies on the uptake and intracellular trafficking of novel cyclodextrin transfection complexes by intestinal epithelial cells

“…Vero African green monkey kidney epithelial cells indicated that CME and CvME are the major routes of uptake for this particular CD (Diaz-Moscoso et al 2010). This is in contrast to the results obtained in the current study and may be explained by structural variations between the CDs and cell type dependent effects.…”

Functional RNA Delivery Targeted to Dendritic Cells By Synthetic Nanoparticles

“…However, these authors did not measure cytosolic delivery, and the pH of 5 required for dissociation is unlikely to be of value for RNA delivery. Polycationic, amphiphilic cyclodextran has been used for DNA delivery [73], although this work focused on epithelial cell lines rather than DC. Dierendonck et alreviewed the work employing hydrogen bonded capsules assembled using poly(methacrylic acid) and degradable dextran sulfate/poly-l-arginine; reductionsensitive linkages were incorporated into the polymers, without introducing polycations [74].…”

Scaffold‐Based microRNA Therapies in Regenerative Medicine and Cancer

“…[78] This work demonstrated a macropinocytosis-mediated uptake mechanism for the dendrimer-miRNA complexes, with substantially improved efficiency in comparison to the original PAMAM formulations reported. [79] Similarly, cyclodextrins undergo unspecific (clathrin independent) uptake [80] but also serve to improve water-solubility and biodistribution of the cargo due to their cyclic oligosaccharide nature. [81] In the case of CPPs, uptake may occur either through transitory micelle formation, endocytosis in its four variants, or direct penetration driven by penetratin or TAT (GRKKR-RQRRR) domains, [82] which explains their documented high transfection efficiencies.…”