Uptake of ricinB-quantum dot nanoparticles by a macropinocytosis-like mechanism
© Iversen et al.; licensee BioMed Central Ltd. 2012
Received: 18 May 2012
Accepted: 23 July 2012
Published: 31 July 2012
There is a huge effort in developing ligand-mediated targeting of nanoparticles to diseased cells and tissue. The plant toxin ricin has been shown to enter cells by utilizing both dynamin-dependent and -independent endocytic pathways. Thus, it is a representative ligand for addressing the important issue of whether even a relatively small ligand-nanoparticle conjugate can gain access to the same endocytic pathways as the free ligand.
Here we present a systematic study concerning the internalization mechanism of ricinB:Quantum dot (QD) nanoparticle conjugates in HeLa cells. Contrary to uptake of ricin itself, we found that internalization of ricinB:QDs was inhibited in HeLa cells expressing dominant-negative dynamin. Both clathrin-, Rho-dependent uptake as well as a specific form of macropinocytosis involve dynamin. However, the ricinB:QD uptake was not affected by siRNA-mediated knockdown of clathrin or inhibition of Rho-dependent uptake caused by treating cells with the Clostridium C3 transferase. RicinB:QD uptake was significantly reduced by cholesterol depletion with methyl-β-cyclodextrin and by inhibitors of actin polymerization such as cytochalasin D. Finally, we found that uptake of ricinB:QDs was blocked by the amiloride analog EIPA, an inhibitor of macropinocytosis. Upon entry, the ricinB:QDs co-localized with dextran, a marker for fluid-phase uptake. Thus, internalization of ricinB:QDs in HeLa cells critically relies on a dynamin-dependent macropinocytosis-like mechanism.
Our results demonstrate that internalization of a ligand-nanoparticle conjugate can be dependent on other endocytic mechanisms than those used by the free ligand, highlighting the challenges of using ligand-mediated targeting of nanoparticles-based drug delivery vehicles to cells of diseased tissues.
Nanomedicine is an interdisciplinary field of research focusing on the development of nanoparticles (NPs) for clinical use in targeted drug delivery and diagnostic in vivo imaging. The goal will often be to increase the efficacy of drugs/ siRNAs at the target tissue and reduce the dose of drug into bystander tissue, and/or to develop NPs into diagnostic imaging agents specifically targeting tumors and diseased tissues. However, studies to fundamentally understand the mechanisms of cell-nanoparticle interactions are still lacking. Investigating whether the nanoparticles themselves might have adverse effects is also of crucial importance. The small sizes of nanoparticles enable them to cross various biological barriers of the body and also to enter the endocytic pathways of the cells, which in turn can give rise to unexpected toxicities. In a previous study, we demonstrated that cellular uptake of quantum dot (QD) nanocrystals that were surface modified with the targeting ligands transferrin (Tf) and ricin, perturbed normal intracellular trafficking in cells [1, 2].
There are multiple types of endocytic pathways distinguished by specific molecular regulators. The clathrin-mediated endocytosis is by far the best studied of these mechanisms and was for a long time believed to be the only endocytic mechanism in addition to phagocytosis and macropinocytosis. However, several clathrin-independent mechanisms have been described, including dynamin-dependent mechanisms such as the RhoA- and caveolae-dependent, and dynamin-independent mechanisms such as the Cdc42-dependent and Arf6-dependent [3, 4]. Dynamin is a large GTPase that mediates vesicle formation by its ability to tubulate and constrict membranes . Caveolae-mediated uptake has been among the most studied routes of dynamin and cholesterol dependent endocytosis. In many studies uptake of nanoparticles (NPs) has been reported to occur via caveolae-mediated endocytosis merely based upon inhibited uptake by the pharmacological inhibitor methyl-β-cyclodextrin (mβCD). Notably, depleting the cell of cholesterol using mβCD also inhibits other endocytic mechanisms, such as clathrin-mediated endocytosis, phagocytosis and macropinocytosis [6, 7]. Moreover, caveolae with a diameter of only 50–100 nm are clearly too small to be responsible for uptake of NPs larger than 100 nm. Caveolae are present in most vascular endothelia playing an important role in transcytosis of blood-borne molecules across the vascular endothelial cell layer, and transcytosis of 10–15 nm gold NPs linked with a caveolae-targeting ligand has been shown . The belief that internalization via caveolae would spare its cargo from being degraded in lysosomes has also been a reason for ‘targeting’ NPs to caveolae. However, the previous model of caveolae giving rise to neutral “caveosomes” has now been revised: The caveosomes are artefacts obtained by overexpression of caveolin-1, and a ligand taken up by caveolae will enter endosomes and be transported to lysosomes .
Although, macropinocytosis in general has been considered to be a dynamin-independent mechanism, the ‘circular dorsal ruffle’-type of macropinocytosis might involve dynamin . Macropinocytosis can in addition to fluid-phase uptake also accommodate uptake of particulate matter such as viruses, bacteria and nanoparticles [11, 12]. Interestingly, dynamin-dependent and amiloride-sensitive macropinocytosis-like mechanisms have been reported for the uptake of bluetongue virus-1 and the Ebola virus [13, 14]. In endothelial cells, multimeric antibody-nanoparticle conjugates directed against the intercellular adhesion molecule (ICAM-1) trigger internalization of large (diameter, 100–400 nm) anti-ICAM-1 and anti-PECAM-1 nanoconjugates by a macropinocytosis-like mechanism that is dynamin-dependent and also requires RhoA activation and actin reorganization [15, 16]. In a recent study, it has been shown that endocytosis of chemokines in endothelial cell lines occurred via a macropinocytosis-like process that was not blocked by siRNA knock-down of PAK1 and CtBP1, two effector proteins of the Rho family GTPase Rac1 . Furthermore, it has been found that specific splice-variants of dynamin-2 were required for the internalization of fluid by endocytic pathways distinct from macropinocytosis .
Toxins such as the plant toxin ricin have for many years been used as valuable tools to study intracellular transport routes in cells, and also uptake of ricin coupled to small gold NPs has been shown in Vero cells [19–22]. Ricin consists of two polypeptide moieties linked by a disulfide bond. The B-moiety binds to glycolipids and glycoproteins with terminal galactose, and can therefore be used as a membrane marker. Studies of ricin endocytosis after inhibition of clathrin-dependent endocytosis by different methods demonstrated that ricin was still endocytosed, and these studies provided some of the first evidence for clathrin-independent endocytosis [23, 24]. Moreover, ricin was still endocytosed after overexpression of the dominant negative mutant dynamin (dyn K44A/G273D) , which inhibited clathrin-mediated endocytosis of both transferrin (Tf) and epidermal growth factor (EGF), whereas fluid phase uptake of horse raddish peroxidase was unaffected . Thus, ricin can be internalized by clathrin and dynamin independent mechanisms in HeLa cells. These endocytic mechanisms still remain incompletely characterized, but recently some of the proteins involved have been identified (for review see ). In a previous study, we found the internalized ricin:QD NPs localizing to the same early and late endosomes as ricin itself, but in contrast to ricin which is also transported to the Golgi apparatus, Golgi transport of the ricin:QD conjugate could not be observed . Recently, a few other studies also revealed a change in ligand behavior after conjugation to NPs: It has been demonstrated in pancreatic cancer cell lines that anti-EGFR antibody-gold nanoparticle conjugates used different and faster endocytosis mechanisms than the anti-EGFR antibody itself , and the valency of TatP domains conjugated to QDs affected the fate of the NPs . Furthermore, multivalent binding of antibodies and ligands of the PECAM-1 glycoprotein to NPs has been shown to trigger internalization of the antibody-ligand nanoconjugates in endothelial cells although the antibodies themselves were not internalized .
In this study we investigated by which endocytic mechanisms small (30 nm) ricinB:QD NP conjugates were internalized, and identified a dynamin-dependent macropinocytosis-like mechanism to be critically involved.
Results and discussion
Here, we have investigated the endocytic mechanisms responsible for internalization of ricinB:QDs, consisting of PEGylated streptavidin-coupled QDs that were conjugated with biotinylated ricinB (multivalent, molar ratio ricinB/QDs of 5), in HeLa cells. The size (hydrodynamic diam.) of the ricinB:QDs conjugates were measured by the Zetasizer to be 30 nm (5 nm variation between batches), small enough to be endocytosed by most endocytic mechanisms.
Internalization of ricinB:QDs is mediated by dynamin-dependent endocytosis that is independent of clathrin
In earlier studies, it has been shown that overexpression of mutant dynamin induced other compensatory dynamin-independent endocytic mechanisms that acted with similar kinetics in uptake of cargo such as ricin and the fluid phase marker HRP [25, 31]. However, the ricinB:QDs can obviously not be internalized efficiently via this compensatory mechanism.
Cholesterol-dependent internalization of RicinB:QDs is independent of RhoA
Effects of actin inhibitors on the ricinB:QD endocytosis
In contrast to our findings, endocytosis of small particles such as QDs (<100 nm) have been reported to be insensitive to cytD in mammalian macrophages, leukocytes and dendritic cells [38, 39]. However, disruption of F actin by cytD has been reported to play a variable role in endocytosis of Tf depending on both the cell line and whether the cells were grown in suspension or not [40, 41]. Recently, is has been shown that membrane tension can decide whether actin is required for clathrin-dependent endocytosis .
Internalization of ricinB:QDs is mediated by a macropinocytosis-like mechanism
The activation of PI3K and the engagement of signaling molecules including Rac1, Arf6 and the RhoA GTPase are common to a variety of actin-dependent processes such as phagocytosis and macropinocytosis . In this context, it is interesting to note that in this study uptake of ricinB: QDs were not inhibited by treatment of the HeLa cells with the inhibitor of Rho, C3 transferase (see Figure 3). Furthermore, overexpression of the dominant-negative mutant Arf6 T27N in a stably transfected HeLa cell line  did not inhibit endocytosis of ricinB: QDs (data not shown). Macropinocytosis has been considered as a regulated form of endocytosis induced in response to growth factors such as epidermal growth factor . Therefore, we would like to know whether the ricinB: QDs could induce such a fluid phase pathway. Using the Olympus ScanR fluorescence microscope, we quantified the uptake of fluorescent dextran-alexa594 in HeLa cells when taken up alone or when co-internalized with the ricinB:QDs for 30 min. No differences in the fluorescence intensities of internalized dextran were measured between the two conditions (data not shown), indicating that uptake of the ricinB:QDs did not significantly change the fluid uptake of dextran during this time period.
Reagents used in this study included the amiloride analoge EIPA (Sigma), cytochalasin D (Sigma), methyl-β-cyclodextrin (Sigma), ADP-ribosyltranferase C3 (Sigma). RicinB subunit was purchased from Vector Laboratories (Burlingame, CA). Quantum dots 655 nm, alexa-555 transferrin (Tf), alexa-594 dextran, alexa-647 phalloidin (Ph), and Hoechst 33342 were purchased from Invitrogen (Carlsbad, CA). The following antibodies were used: Rabbit anti-EEA1 (Cell Signaling Technologies), mouse anti-CD63 (Developmental Studies Hybridoma Bank, Univ. of Iowa). The Cy2- and Cy3-labeled secondary antibody conjugates of donkey anti-rabbit and donkey anti-mouse were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA, USA).
Biotinylation of ricinB
RicinB (1 mg/ml) was biotinylated according to manufacturer’s instructions using NHS-SS-biotin (Pierce, Rockford, IL) in a 1:20 molar ratio of ricin:biotin. The reaction took place at room temperature in the dark for 45 min. Unincorporated NHS-SS-biotin was removed using a spin column (Micron, YM-10, Millipore).
Hydrodynamic size measurement
The hydrodynamic diameter of the streptavidin-coupled QD655 particles was measured in PBS buffer or in cell culture medium using using a Zetasizer Nano ZS (Malvern Instruments Ltd, Worcestershire, UK).
HeLa cells were maintained at 37°C and 5% CO2 in Dulbecco’s Modified Eagle medium, DMEM (Invitrogen, Carlsbad, CA, USA), supplemented with 10% v/v fetal bovine serum (PAA Laboratories, Linz, Austria), 100 U/ml penicillin (Invitrogen) and 100 μg/ml streptomycin (Invitrogen).
Cellular uptake of ricinB:qdot nanoconjugates by confocal fluorescence microscopy
HeLa cells or HeLa Dyn-K44A cells (seeded 2x104cells/well in 24-well trays) were cultured on coverslips for 24 h or 48 h prior to the experiments, respectively. Then, ricinB:QDs conjugates were prepared directly at the cell surface, as previously described: Cells were washed in cold Hepes-medium prior to incubation with the biotinylated ricinB (200 nM) ligand in Hepes medium for 10 min on ice. The cells were briefly washed 2x in tetra-borate buffer (50 mM sodium-borate, pH8.3; 215 mM sucrose) and then incubated with the streptavidin-coupled QD655 (20 nM) in tetra-borate buffer for 5 min on ice . Subsequently, the cells were washed 2x in Hepes medium before endocytosis of the ricinB:QDs (molar ratio, 5:1) was performed by incubating the cells in Hepes medium at 37C for various times with or without the appropriate inhibitors. The ricinB:QDs were also co-internalized with other ligands such as Tf-alexa-555 and dextran-alexa-647.
After fixation in 10% (w/v) formalin for 15 min, the cells were permeabilized and blocked in 0.1% Triton X-100 and 1% BSA in PBS for 1 hour at room temperature. The cells were immuno-stained with the following primary antibodies: mouse anti-EEA1 ab. (1:100) and mouse anti-CD63 ab. (1:200). The secondary antibody-fluorescent dye conjugates used were: Donkey anti-rabbit-Cy2 (1:200), donkey anti-rabbit-Cy3 (1:500) and donkey anti-mouse-Cy2 (1:500). Coverslips were mounted in Mowiol (Calbiochem) and examined using a confocal microscope (LSM 788; Carl Zeiss MicroImaging, Inc.) equipped with a Neo-Fluar 63x/1.45 oil immersion objective. Image processing and analysis were done with Zeiss LSM 510 software version 3.2 and Adobe Photoshop 7.0.
Vector-based siRNA knock-down of clathrin
Clathrin heavy-chain was knocked down with a vector-based siRNA construct . Cells were transiently transfected for 3 days with plasmid DNA using Fugene 6 (Roche Diagnostics, Mannheim, Germany), according to the manufacturer’s protocol.
Tore-Geir Iversen and Nadine Frerker were the recipients of career and post-doctoral fellowships, respectively, from the FUGE and NANOMAT programmes. This work was supported by the Research Council of Norway, the Norwegian Cancer society and Helse Sør-Øst.
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