Dries Vercauteren

The Use of Inhibitors to Study Endocytic Pathways of Gene Carriers: Optimization and Pitfalls

Nonviral gene complexes can enter mammalian cells through different endocytic pathways. For efficient optimization of the gene carrier it is important to profile its cellular uptake, because this largely determines its intracellular processing and subsequent transfection efficiency. Most of the current information on uptake of these gene-delivery vehicles is based on data following the use of chemical inhibitors of endocytic pathways. Here, we have performed a detailed characterization of four commonly used endocytosis inhibitors [chlorpromazine, genistein, methyl-beta-cyclodextrin (MbetaCD), and potassium depletion] on cell viability and endocytosis in five well-described cell lines. We found that chlorpromazine and to a lesser extent MbetaCD significantly decreased cell viability of some cell lines even after short incubation periods and at concentrations that are routinely used to inhibit endocytosis. Through analyzing the uptake and subcellular distribution of two fluorescent endocytic probes transferrin and lactosylceramide (LacCer) that are reported to enter cells via clathrin-dependent (CDE) and clathrin-independent (CIE) mechanisms, respectively, we showed poor specificity of these agents for inhibiting distinct endocytic pathways. Finally, we demonstrate that any inhibitory effects are highly cell line dependent. Overall, the data question the significance of performing endocytosis studies with these agents in the absence of very stringent controls.

On the cellular processing of non-viral nanomedicines for nucleic acid delivery: Mechanisms and methods

In the field of nanomedicine, ample attention has been paid to the development of nanocarriers for the intracellular delivery of therapeutic cargo, such as nucleic acids for gene therapy. The efficiency with which these non-viral carriers deliver their payload at the required intracellular site of action remains low. Despite extensive research on cellular attachment, endocytosis and intracellular trafficking of nanocarriers, clear-cut rules for the design of effective nanocarriers to improve nucleic acid transfer are still lacking. This is mainly caused by the cell type-dependence of this highly dynamic cellular processing, and to the lack of reliable methods to study these events. For these reasons there is a strong demand for the development and standardization of such methods in order to better understand the intracellular dynamics of nanomedicine processing and validate cellular and intracellular targeting strategies. This review aims at providing an overview of the different processes that are currently known to be involved in the cellular processing of nanomedicines, with a focus on cellular internalization mechanisms, as this has received a great deal of attention in the last couple of years. Furthermore, the intracellular hurdles that need to be overcome to allow efficient NA transfer will be critically discussed. In addition, an overview will be given of various methodologies that have been applied to unravel these cellular processing mechanisms, with a discussion on their strengths and weaknesses.

The role of nanoparticle concentration-dependent induction of cellular stress in the internalization of non-toxic cationic magnetoliposomes.

Magnetoliposomes (MLs), built up of ultrasmall iron oxide cores each individually surrounded by a lipid bilayer, have emerged as highly biocompatible nanoparticles and promising tools in many biomedical applications. To improve cell uptake, cationic amphiphiles are inserted into the ML coat, but this often induces cytotoxic effects. In the present work, we synthesized and tested a cationic peptide-lipid conjugate (dipalmitoylphosphatidylethanolamine-succinyl-tetralysine [DPPE-succ-(Lys)4]) which is entirely composed of biodegradable moieties and specifically designed to exert minimal cytotoxic effects. Uptake studies with both murine 3T3 fibroblasts and C17.2 neural progenitor cells shows 95.63 +/- 5.83 pg Fe and 87.46 +/- 5.62 pg Fe per cell after 24 h, respectively, for 16.66% DPPE-succ-(Lys)4-containing MLs, with no effect on cell viability. However, these high intracellular nanoparticle concentrations transiently affect actin cytoskeleton architecture, formation of focal adhesion complexes and cell proliferation, returning to control levels after approximately 7 days post ML-incubation in both cell types. This study points out the great need for thorough characterization of cell-nanoparticle interactions as subtle time-dependent effects are hard to monitor and commonly used viability and functionality assays are not sufficient to address the broad spectrum of possible interferences of the nanoparticle with normal cell functioning.

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

It 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 (<70nm). Since paCDs resemble both cationic polymers and cationic lipids, it is conceivable that the corresponding pDNA-paCD nanoparticles (CDplexes) might use the cell internalization 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 beta-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.

Dynamic Colocalization Microscopy To Characterize Intracellular Trafficking of Nanomedicines

To gain a better understanding of intracellular processing of nanomedicines, we employed quantitative live-cell fluorescence colocalization microscopy to study endosomal trafficking of polyplexes in retinal pigment epithelium cells. A new, dynamic colocalization algorithm was developed, based on particle tracking and trajectory correlation, allowing for spatiotemporal characterization of internalized polyplexes in comparison with endosomal compartments labeled with EGFP constructs. This revealed early trafficking of the polyplexes specifically to Rab5- and flotillin-2-positive vesicles and subsequent delivery to Rab7 and LAMP1-labeled late endolysosomes where the major fraction of the polyplexes remains entrapped for days, suggesting the functional loss of these nanomedicines. Colocalization of polyplexes with the autophagy marker LC3 suggests for the first time that the process of xenophagy could play an important role in the persistent endosomal entrapment of nanomedicines.

Flotillin-dependent endocytosis and a phagocytosis-like mechanism for cellular internalization of disulfide-based poly(amido amine)/DNA polyplexes

Extensive research is currently performed on designing safe and efficient non-viral carriers for gene delivery. To increase their efficiency, it is essential to have a thorough understanding of the mechanisms involved in cellular attachment, internalization and intracellular processing in target cells. In this work, we studied in vitro the cellular dynamics of polyplexes, composed of a newly developed bioreducible poly(amido amine) carrier, formed by polyaddition of N,N-cystamine bisacrylamide and 1-amino-4-butanol (p(CBA-ABOL)) on retinal pigment epithelium (RPE) cells, which are attractive targets for ocular gene therapy. We show that these net cationic p(CBA-ABOL)/DNA polyplexes require a charge-mediated attachment to the sulfate groups of cell surface heparan sulfate proteoglycans in order to be efficiently internalized. Secondly, we assessed the involvement of defined endocytic pathways in the internalization of the polyplexes in ARPE-19 cells by using a combination of endocytic inhibitors, RNAi depletion of endocytic proteins and live cell fluorescence colocalization microscopy. We found that the p(CBA-ABOL) polyplexes enter RPE cells both via flotillin-dependent endocytosis and a PAK1 dependent phagocytosis-like mechanism. The capacity of polyplexes to transfect cells was, however, primarily dependent on a flotillin-1-dependent endocytosis pathway.

siRNA and pharmacological inhibition of endocytic pathways to characterize the differential role of macropinocytosis and the actin cytoskeleton on cellular uptake of dextran and cationic cell penetrating peptides octaarginine (R8) and HIV-Tat

Cell penetrating peptides (CPPs) have been extensively studied as vectors for cellular delivery of therapeutic macromolecules. It is widely accepted that they can enter cells directly across the plasma membrane but also gain access through endocytic pathways that are yet to be fully defined. Here we developed siRNA methods in epithelial cell lines, HeLa and A431, to inhibit endocytic pathways regulated by clathrin heavy chain, flotillin-1, caveolin-1, dynamin-2 and Pak-1. In each case, functional uptake assays were developed to characterize the requirement for these proteins, and the pathways they regulate, in the internalisation of defined endocytic probes and also the CPPs octaarginine and HIV-Tat. Peptide uptake was only inhibited in A431 cells depleted of the macropinocytosis regulator Pak-1, but experimental variables including choice of cell line, pharmacological inhibitor, macropinocytic probe and serum starvation significantly influence our ability to assess and assign this pathway as an important route for CPP uptake. Actin disruption with Cytochalasin D inhibited peptide entry in both cell lines but the effects of this agent on dextran uptake was cell line dependent, reducing uptake in HeLa cells and increasing uptake in A431 cells. This was further supported in experiments inducing actin stabilisation by Jasplakinolide, emphasising that the actin cytoskeleton can both promote and hinder endocytosis. Overall the data identify important aspects regarding the comparative mechanisms of CPP uptake and macropinocytosis, and accentuate the significant methodological challenges of studying this pathway as an endocytic portal and an entry route for drug delivery vectors.

A New FRAP/FRAPa Method for Three-Dimensional Diffusion Measurements Based on Multiphoton Excitation Microscopy

We present a new convenient method for quantitative three-dimensionally resolved diffusion measurements based on the photobleaching (FRAP) or photoactivation (FRAPa) of a disk-shaped area by the scanning laser beam of a multiphoton microscope. Contrary to previously reported spot-photobleaching protocols, this method has the advantage of full scalability of the size of the photobleached area and thus the range of diffusion coefficients, which can be measured conveniently. The method is compatible with low as well as high numerical aperture objective lenses, allowing us to perform quantitative diffusion measurements in three-dimensional extended samples as well as in very small volumes, such as cell nuclei. Furthermore, by photobleaching/photoactivating a large area, diffusion along the optical axis can be measured separately, which is convenient when studying anisotropic diffusion. First, we show the rigorous mathematical derivation of the model, leading to a closed-form formula describing the fluorescence recovery/redistribution phase. Next, the ability of the multiphoton FRAP method to correctly measure absolute diffusion coefficients is tested thoroughly on many test solutions of FITC-dextrans covering a wide range of diffusion coefficients. The same is done for the FRAPa method on a series of photoactivatable green fluorescent protein solutions with different viscosities. Finally, we apply the method to photoactivatable green fluorescent protein diffusing freely in the nucleus of living NIH-3T3 mouse embryo fibroblasts.

Stable long-term intracellular labelling with fluorescently tagged cationic magnetoliposomes

Iron oxide nanocrystals that are dextran coated are widely exploited biomedically for magnetic resonance imaging (MRI), hyperthermia cancer treatment and drug or gene delivery. In this study, the use of an alternative coating consisting of a phospholipid bilayer directly attached to the magnetite core is described. The flexible nature of the magnetoliposome (ML) coat, together with the simple production procedure, allows rapid and easy modification of the coating, offering many exciting possibilities for the use of these particles in biomedical applications. Upon incubation of neutral MLs with an equimolar amount of cationic 1,2‐distearoyl‐3‐trimethylammoniumpropane (DSTAP)‐bearing vesicles, approximately one third of the cationic lipids are incorporated into the ML coat. This is in line with a theoretical model predicting transferability of only the outer leaflet phospholipids of bilayer structures. Most interestingly, the use of MLs containing 3.33 % DSTAP with a positive ζ‐potential of (31.3±7.3) mV (mean ±SD) at neutral pH, results in very heavy labelling of a variety of biological cells (up to (70.39±4.52) pg of Fe per cell, depending on the cell type) without cytotoxic effects. The results suggest the general applicability of these bionanocolloids for cell labelling. Mechanistically, the nanoparticles are primarily taken up by clathrin‐mediated endocytosis and follow the endosomal pathway. The fate of the ML coat after internalisation has been studied with different fluorescent lipid conjugates, which because of the unique features of the ML coat can be differentially incorporated in either the inner or the outer layer of the ML bilayer. It is shown that, ultimately, iron oxide cores surrounded by an intact lipid bilayer appear in endosomal structures. Once internalised, MLs are not actively exocytosed and remain within the cell. The lack of exocytosis and the very high initial loading of the cells by MLs result in a highly persistent label, which can be detected, even in highly proliferative 3T3 fibroblasts, for up to at least one month (equivalent to approximately 30 cell doublings), which by far exceeds any values reported in the literature.

Advanced fluorescence microscopy methods illuminate the transfection pathway of nucleic acid nanoparticles

A great deal of attention in biopharmacy and pharmaceutical technology is going to the development of nanoscopic particles to efficiently deliver nucleic acids to target cells. Despite the great potential of nucleic acids for treatment of various diseases, progress in the field is fairly slow. One of the causes is that development of suitable nanoscopic delivery vehicles is hampered by insufficient knowledge of their physicochemical and biophysical properties during the various phases of the transfection process. To address this issue, in the past decade we have developed and applied advanced fluorescence microscopy techniques that can provide a better insight in the transport and stability of nanoparticles in various biological media. This mini-review discusses the basic principles of fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS) and single particle tracking (SPT), and gives an overview of studies in which we have employed these techniques to characterize the transport and stability of nucleic acid containing nanoparticles in extracellular media and in living cells.