Internalization of mRNA Lipoplexes by Dendritic Cells - Molecular

Aug 15, 2012 - Virology Unit, Department of Biomedical Sciences, Institute of Tropical Medicine of Antwerp, Nationalestraat 155, Antwerp, Belgium...
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Internalization of mRNA Lipoplexes by Dendritic Cells Winni De Haes,*,† Greet Van Mol,† Céline Merlin,† Stefaan C. De Smedt,‡ Guido Vanham,†,§,∥ and Joanna Rejman‡ †

Virology Unit, Department of Biomedical Sciences, Institute of Tropical Medicine of Antwerp, Nationalestraat 155, Antwerp, Belgium ‡ Laboratory of General Biochemistry and Physical Pharmacy, Ghent University, Harelbekestraat 72, Ghent, Belgium § Faculty of Pharmaceutical, Veterinary and Biomedical Sciences, University of Antwerp, Belgium ∥ Faculty of Medicine and Pharmacology, Vrije Universiteit Brussel, Belgium ABSTRACT: Lipoplexes, composed of Lipofectamine and mRNA encoding HIV Gag protein, were shown to be internalized by dendritic cells (DCs) and promote antigen presentation to stimulate HIV-specific T cell responses. Using confocal microscopy, we showed that one-third of fluorescently labeled mRNA containing lipoplexes are colocalized with late endosomes. We further investigated the effect of inhibitors, blocking phagocytosis, macropinocytosis, and clathrin- and caveolae-mediated endocytosis, on both the internalization of the lipoplexes by DCs and the transfection efficiency. We observed that chloropromazine had no effect on the cellular uptake or transfection efficiency, excluding the involvement of clathrin-mediated endocytosis. Cytochalasin D, inhibiting macropinocytosis and phagocystosis, strongly reduced internalization (50%) of the lipoplexes as well as protein expression (70%). Amiloride, which should specifically block macropinocytosis, induced only a modest reduction of uptake and transfection. Genistein and dynasore induced a strong reduction of on the level of protein expression (>70%), but not the overall uptake. Our results indicate that transfectioneffective mRNA lipoplex internalization by DCs, i.e., uptake that results in protein expression, preferentially proceeds by macropinocytosis and/or phagocytosis. KEYWORDS: Lipofectamine, mRNA, endocytosis, micropinocytosis, phagocytosis, dendritic cells



extent as mRNA-electroporated DCs.6 To further improve this system, it is important to better understand the pathways of lipoplex internalization that lead to productive transfection in DCs. Mammalian cells internalize extracellular macromolecules by endocytosis, which can be divided into two categories: phagocytosis, restricted to specialized cells such as macrophages, monocytes, neutrophils and DCs, and pinocytosis, operating in all cell types.7 Pinocytosis can be further divided into macropinocytosis, clathrin-mediated endocytosis (CME) and clathrin-independent endocytosis. The latter can be subdivided into (1) caveolae-mediated dynamin-dependent endocytosis (cavME), (2) flotillin-mediated dynamin-independent endocytosis, (3) ARF6-mediated endocytosis and (4) CLIC/GEEC (clathrin-independent carriers/GPI-enriched endosomal compartment).8 It is believed that such a diversity of endocytosis pathways is employed by the cells to perform different tasks.

INTRODUCTION Dendritic cells (DCs) play a key role in the development of immunotherapies against cancer and infectious diseases, including HIV. As sentinels of the immune system, they take up and process antigens to present them on MHC class I and II molecules to T cells. Ex vivo electroporation of DCs with mRNAs, encoding HIV-1 proteins covering a wide range of quasi species, has been demonstrated to be an interesting strategy to induce HIV-1 specific T cell responses both in vitro1,2 and in vivo.3−5 This approach, however, is time-consuming, labor intensive and thus not applicable to a wide range of patients. Moreover, it implies the use of sophisticated technology and therefore it is not readily implementable in less developed countries. Therefore, there is a need to design novel delivery strategies that could allow direct in vivo loading of DCs. One possibility is to employ nonviral carriers that could deliver antigens to DCs in vivo. As compared to viral carriers, they are easier to prepare, more versatile and mostly not immunogenic by themselves. Previously we have shown that lipoplexes consisting of mRNA encoding HIV Gag protein (mGag) and Lipofectamine 2000, being a cationic lipid, transfect DCs in vitro with high efficiencies. Importantly, the transfected DCs were able to expand HIV-1 specific CD4+ and CD8+ T cells, to the same © 2012 American Chemical Society

Received: Revised: Accepted: Published: 2942

June 18, 2012 August 6, 2012 August 15, 2012 August 15, 2012 dx.doi.org/10.1021/mp3003336 | Mol. Pharmaceutics 2012, 9, 2942−2949

Molecular Pharmaceutics

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Most endocytosis pathways lead to formation of vesicle-like structures that fuse with early/sorting endosomes. The luminal pH of these structures is around 6. It is generally accepted that intracellular trafficking from this compartment is determined by the vesicle origin and the cargo carried inside. Early endosomes can be trafficked directly or via recycling endosomes to the plasma membrane. Alternatively, they can further mature into late endosomes. This process is characterized by a further drop in the luminal pH (>5) and formation of multivesicular bodies that facilitate sorting of membrane-associated receptors.9 Late endosomes can fuse with lysosomes and this way expose their cargo to the action of more than 40 different hydrolases. It is conceivable, therefore, that nucleic acid complexes, depending on the nature of the endosomal compartment where they end up, may either become or not become biologically active. For instance, particles taken up by CME end up in the lysosomal compartment, which eventually leads to their degradation.9 This implies that transfection can only be successful if particles carrying nucleic acids timely escape from preacidic compartments and release their cargo in the cytoplasm. Therefore, various cationic polymers and lipids have been developed to favor escape from the endosomal compartment by influencing the osmotic pressure inside the endosomes10 or by destabilizing the endosomal membrane.11 In general, the uptake pathway is determined by the chemical structure of the particles (size, charge, stability) as well as the cell type and its metabolism.12 There is no consensus with respect to the question which endocytosis pathway is most favorable for nucleic acid delivery. Some reports indicate that CME leads to more effective transfection than clathrin-independent endocytosis.12 Others found that cavME results in optimal transfection since it does not involve acidic intracellular compartments and thus prevents acidic degradation.13,14 Not much has been published on uptake and intracellular trafficking of mRNA complexes. Therefore, in the present study we used a set of chemical endocytosis inhibitors to investigate the pathways used by monocyte-derived DCs to internalize Lipofectamine−mRNA complexes. The effect of these inhibitors on expression of the encoded protein was also evaluated. This allowed better understanding of the correlation between the mechanism of uptake and transfection efficiency of lipoplexes, which to our knowledge has not been reported so far. Since all endocytic pathways are active in dendritic cells, these cells present an optimal tool to study mechanisms involved in the particle uptake. An important aspect of the study is the fact that the experiments were performed on primary cells which present a more clinically relevant model than stable cell lines.

The original humanized cDNA was kindly provided by Bernard Verrier (Biomérieux, Lyon, France). Both plasmids were propagated in Escherichia coli supercompetent cells (Stratagene, La Jolla, CA, USA) and purified on endotoxin-free QIAGENtip 500 columns (Qiagen, Chatsworth, CA, USA). The plasmids were linearized with SpeI restriction enzyme (MBI Fermentas, St. Leon-Rot, Germany), purified using a PCR purification kit (Qiagen, Venlo, The Netherlands) and used as DNA templates for in vitro transcription. Transcription was carried out in a final 20−200 μL reaction mix at 37 °C using the T7MessageMachine Kit (Ambion, Austin, TX, USA) to generate 5′ capped in vitro transcribed (IVT) mRNA. Purification of mRNA was performed by DNase I digestion followed by LiCl precipitation, according to the manufacturer’s instructions. RNA concentration was assayed by spectrophotometric analysis at OD260. mRNA was stored in RNase free water at −80 °C. Fluorescently Labeled mRNA. mRNA was labeled with Cy5 using an ULS mRNA Fluorescent Labeling Kit (Kreatech, Amsterdam, The Netherlands) according to the manufacturer’s instructions with some minor modifications. Briefly, 2 μg of mRNA, 6 μL of nuclease-free water, 2 μL of Cy5 probe and 2 μL of buffer solution (Kreatech kit) were mixed. The mixture was incubated at 85 °C in a water bath for 15 min and then applied on a Kreatech column, and labeled mRNA was eluted with 100 μL of nuclease-free water. After centrifugation at 14 000 rpm for 15 min at 4 °C, the supernatant was discarded and the pellet resuspended with 10 μL of nuclease-free water. mRNA labeled this way was used for microscopy studies. To label gag mRNA with YOYO-1 (Invitrogen, Merelbeke, Belgium), 20 μg of mRNA was first diluted in 100 μL of RNasefree water. This solution was added to the staining solution containing 3 μL of YOYO-1 in RNase-free water (300 μL). The mixture was incubated for 1 h at RT. Subsequently, 40 μL of LiCl (the T7MessageMachine Kit, Ambion) and 800 μL of ice cold ethanol (100%) were added and the mixture was incubated for 30 min at −80 °C. mRNA was pelleted by centrifugation at 14 000 rpm for 15 min and resuspended in 20 μL of RNase-free water. mRNA labeled this way was used for microscopy studies and flow cytometry. Lipoplex Preparation. Lipoplexes were prepared in serumfree RPMI medium (Lonza, Breda, The Netherlands) by mixing mRNA and Lipofectamine 2000 (Invitrogen, Merelbeke, Belgium) at a 1:1 ratio.6 Briefly, 10 μg of mRNA and 10 μL of Lipofectamine (1 mg/mL) were diluted separately in 250 μL of serum-free RPMI medium. The Lipofectamine solution was added to the mRNA solution and incubated for 15 min at RT. Lipoplexes were stored at 4 °C for maximally 4 h before transfection. Size Determination. The average particle size of the mRNA complexes was determined by dynamic light scattering using the Zetasizer Nano ZS (Malvern, Worcestershire, U.K.). Lipoplexes were prepared in serum-free RPMI medium (Lonza) as described above. The measurements were performed at 37 °C. The size of freshly prepared unlabeled lipoplexes was 731 (±171) nm, YOYO-1-labeled lipoplexes was 719 (±124) nm and Cy5-labeled lipoplexes was 762 (±196) nm. Separation of Mononuclear Cell Fractions and in Vitro Generation of Monocyte-Derived DCs. Peripheral blood samples (buffy coats) from healthy donors were kindly provided by the Antwerp Blood Transfusion Center (Red Cross of Flanders, Belgium). Peripheral blood mononuclear



MATERIALS AND METHODS Materials. Chemical inhibitors: amiloride, chlorpromazine, dynasore, genistein, methyl-β-cyclodextrin and methyl palmitate were purchased from Sigma Aldrich (Bornem, Belgium). Cytochalasin D (cyto D), YOYO-1, LysoSensor Green DND189, and Lipofectamine 2000 were purchased from Invitrogen (Merelbeke, Belgium). Production of in Vitro Transcribed mRNA. The pGEM4Z/EGFP/A64 plasmid was kindly provided by Prof. Dr. E. Gilboa (Duke University Medical Center, Durham, NC, USA), and pGEM4Z/hHxB-2-gag/A64 plasmid was generated by the Laboratory of Physiology and Immunology in Brussels. The latter was used to prepare a “humanized” (codonoptimized) mRNA encoding the HxB2 HIV-1 Gag protein.15 2943

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Table 1. Overview of Chemical Inhibitors Used with Their Mode of Action and Applied Concentrations chemical inhibitor genistein dynasore cytochalasin D methyl-βcyclodextrin chlorpromazine amiloride methyl palmitate rottlerina a

range of concns tested

mode of action

appl concn

viability DCs, %

inhibits cavME, raft-dependent endocytosis and macropinocytosis by affecting actin cytoskeleton30 inhibits dynamin23 interferes with actin polymerization, affects phagocytosis and macropinocytosis31,32 removes cholesterol from the cell membrane33

100−600 μM

200 μM

86

60−200 μM 60−200 μM 0.1−10 mM

100 μM 2 μM 0.1 mM

88 87 76

dissociates clathrin and adaptor proteins from the plasma membrane; effects receptor recycling; this way affects CME26 inhibits Na+/H+ ion exchange, membrane ruffling, affects macropinocytosis34 inhibits phagocytosis in Kupffer cells29 inhibits protein kinase C,35 and it is a specific inhibitor of macropinocytosis in dendritic cells36

10−20 μg/mL

10 μg/ mL 20 μM 1 mM 1 μM

80

10−20 μM 0.5−20 mM 1−10 μM

79 78 59a

Due to high toxicity rottlerin was excluded from further studies.

Figure 1. Internalization of mRNA lipoplexes by monocyte-derived dendritic cells. MoDCs were incubated with Lipofectamine−mRNA complexes for 3 h. To visualize the complexes, mRNA was labeled with YOYO-1. Extracellular fluorescence was quenched with trypan blue. (a) A representative confocal microscope image. (b) Flow cytometric analysis. DCs were gated based on FSC and SSC. YOYO-1 expression (FL-1) is shown on the Yaxis versus FSC in the X-axis.

associated fraction of YOYO-1 labeled lipoplexes was quenched with 0.5% trypan blue solution (in PBS, 5 min, RT). After thorough washing, fluorescence was measured with FACScan (BD Bioscience). YOYO-1 was measured in FL-1. To quantify numbers of GFP-positive cells, transfected DCs (1 × 106) were harvested, stained with Via-Probe (BD Biosciences, Erembodegem, Belgium) and analyzed by FACScan (BD Biosciences). DCs were gated, based on their large forward scatter (FSC) and large side scatter (SSC) profile, thus excluding cell debris. Within this “DC gate”, dead cells were excluded using Viaprobe (FL3) and the percentages of GFP-positive living cells were measured in FL1. Data analysis was performed with FlowJo version 8.8.4 (Tree Star Inc., San Carlos, CA). Statistical Analysis. Statistical analysis was performed with GraphPad Prism software (GraphPad Software, Inc., San Diego, CA, USA). A paired Student’s t test was performed to determine statistical significance of the data. P values of ≤0.05 were considered significant (* P < 0.05, **P < 0.01).

cells (PBMC) were isolated using Lymphoprep (Lucron, Sint Martens-Latem, Belgium). Monocytes (Mo) were isolated from PBMC by magnetic isolation using CD14+ microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions with purity of 98%. Mo-derived immature DCs (iDCs) were generated as previously described,16 by supplementing RPMI medium with granulocyte-monocyte colony stimulating factor (GM-CSF), Interleukin-4 (IL-4) in RPMI (Lonza, Breda, The Netherlands) and 2.5% pooled human serum (PHS, PAA laboratories GmbH, Pashing, Austria). Dendritic Cell Transfection and Inhibition Studies. iDCs were harvested six days after their 6 day differentiation and seeded at a density of 2 × 106 in 2 mL of RPMI with 2.5% of PHS in 6-well plates. One hour after seeding, the inhibitors (working concentrations in Table 1) were added and incubated with the cells for 1 h. Then the lipoplexes were added and incubated with the cells for 3 h. Lipoplex uptake or GFP expression was then evaluated as described in the next paragraphs. Confocal Microscopy. The expression of green fluorescent protein in DCs was visualized using a Carl Zeiss LSM 700 laser scanning microscope (Zaventem, Belgium) with a 63 × 1.4 oil objective. Lysosomes were stained with 1 μM LysoSensor Green DND-189 (Invitrogen, Merelbeke, Belgium) added to the cells 30 min before visualization. To acquire confocal images, DCs were seeded on MatTek coverslip (1.5) glass bottom dishes (MatTek Corporation, MA, USA). Flow Cytometry. To quantify lipoplex uptake, DCs (1 × 106) transfected with YOYO-1 labeled mRNA were harvested and washed. The fluorescence of the plasma membrane-



RESULTS AND DISCUSSION Effect of Endocytosis Inhibitors on the Internalization of Lipoplexes by MoDCs. We first studied the internalization of lipoplexes composed of Lipofectamine and mRNA by human monocyte-derived dendritic cells (MoDCs). To visualize complexes, mRNA was labeled with YOYO-1. The evidence of internalization was obtained by confocal microscopy and flow cytometry. To distinguish between actual uptake inside the cell and association with the cell membrane, the extracellular fluorescence was quenched with trypan blue.17,18 Confocal 2944

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Figure 2. Effect of inhibitors on internalization of mRNA lipoplexes by MoDCs. MoDCs were pretreated with inhibitors for 1 h. YOYO-1-labeled mRNA was complexed with Lipofectamine and incubated with the cells in the presence of inhibitors for 3 h. (a) Flow cytometry analysis was applied to quantify lipoplex uptake following transfection with LF−mGag complexes. (b) Both mean fluorescence intensity (MFI, white bars) and percentage of positive cells (green bars) are presented. The values for untreated MoDCs were set as 100%. Extracellular fluorescence was quenched with trypan blue. Mean values ± SD were obtained from six independent experiments. Student’s t test was performed to determine the statistical significance of the data (* P < 0.05; ** P < 0.01).

were incubated with MoDCs in the presence of different inhibitors and the uptake of lipoplexes was assessed by flow cytometry 3 h after adding the complexes to the cells. To discriminate between cell surface-bound and internalized lipoplexes, extracellular fluorescence was quenched by addition of trypan blue. As shown in Figure 2, treatment with all inhibitors, except chlorpromazine, had some effect on the uptake of lipoplexes. While for most inhibitors the effect observed was limited to maximally 30%, cytochalasin D reduced the lipoplex uptake by 50%. These results point to phagocytosis and/or macropinocytosis as major mechanism(s) involved in lipoplex uptake by MoDCs. The lack of inhibition by chlorpromazine suggests that there is no involvement of CME. Colocalization Studies of Lipoplexes and Lysosomes. Most endocytosis pathways lead to the formation of vesicle-like structures that fuse with early/sorting endosomes. It is conceivable that the internalization pathway determines intracellular trafficking and therefore may lead to different transfection efficiencies. To obtain more insight into the intracellular localization of mRNA lipoplexes, MoDCs were incubated with Cy5-labeled mRNA complexed with Lipofectamine. The lysosomal compartment was stained with Lysosensor (pKa 5.2). The samples were analyzed by confocal microscopy. As shown in Figure 3, one-third of the lipoplexes colocalized with the lysosomal compartment. Effect of Endocytosis Inhibitors on Transfection Mediated by Lipofectamine−mRNA Complexes. The

images presented in Figure 1a demonstrate intracellular localization of the lipoplexes. These results were confirmed by flow cytometry. Data presented in Figure 1b demonstrate that almost 50% of DCs internalized the lipoplexes. A panel of chemical inhibitors was selected to study their effect on lipoplex uptake.19,20 Different concentrations of the inhibitors were tested on human MoDCs. Inhibitors that decreased viability of DCs by more than 30% were excluded from the study. From the seven remaining inhibitors, the highest concentration of each inhibitor that did not affect the viability of DCs was chosen for further experiments (Table 1). Methyl-β-cyclodextrin (MβCD), extracting cholesterol from the plasma membrane, is used as a general endocytosis inhibitor.21 Genistein is a tyrosine kinase inhibitor that affects the actin cytoskeleton and prevents recruitment of dynamin. It is essential for caveolae/raft dependent endocytosis and macropinocytosis.22 Dynasore, by inhibiting dynamin, affects clathrin-mediated endocytosis, caveolae-dependent uptake and phagocytosis.23 Cytochalasin D interferes with actin polymerization and inhibits both phagocytosis and macropinocytosis.24,25 Chlorpromazine inhibits formation of clathrin-coated pits and thus interferes with clathrin-mediated endocytosis.26 Amiloride, an inhibitor of macropinocytosis, blocks Na+/H+ ion exchange in the plasma membrane.27,28 Methyl palmitate has been shown to inhibit phagocytosis in Kupffer cells.29 To determine which pathways are involved in the uptake of Lipofectamine−mRNA complexes, YOYO-1 labeled lipoplexes 2945

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results presented in the preceding paragraphs suggest that phagocytosis and/or macropinocytosis are involved in lipoplex uptake and that a significant proportion of the internalized complexes ends up in the lysosomal compartment. Therefore, in the next set of experiments we evaluated which endocytosis pathway leads to productive transfection. To this end MoDCs were transfected with mRNA encoding green fluorescence protein (mGFP) complexed with Lipofectamine. The complexes were incubated with the cells in the presence of the inhibitors. Transfection efficiency was assayed 3 h later since our earlier studies showed that maximal protein expression was achieved at this time point. Both the effect of inhibitors on the percentage of GFP-positive cells and the associated MFI are shown in Figure 4. Transfection efficiency of MoDCs was strongly inhibited by cytochalasin D, an inhibitor of macropinocytosis and phagocytosis. This is compatible with the strong inhibitory effect of this compound on lipoplex uptake as presented in

Figure 3. Colocalization of mRNA lipoplexes and lysosomes in MoDCs. mRNA labeled with Cy5 (red) was complexed with Lipofectamine and incubated with MoDCs for 3 h. Lysosomes were labeled with Lysosensor (green). (a) Colocalization of lipoplexes and lysosomes is seen in yellow. (b) PMT channel. (c) Green channel (lysosomes). (d) Red channel, Cy5 labeled lipoplexes.

Figure 4. Effect of chemical inhibitors on GFP expression in MoDCs. (a) A representative example of the flow cytometric analysis applied to quantify protein expression following transfection with LF−mGFP complexes in the absence or presence of inhibitors. (b) Summary of the effects of endocytosis inhibitors on transfection efficiency. MoDCs were preincubated with the inhibitors for 1 h. Then LF−mGFP complexes were added and incubated with the cells for 3 h. Transfection efficiency was evaluated by flow cytometry. Both mean fluorescence intensity (MFI, white bars) and percentage of positive cells (green bars) are represented. The values obtained for untreated DCs were set as 100%. Mean values ± SD were obtained from four independent experiments. Student’s t test was performed to determine the statistical significance of the data (* P < 0.05; ** P < 0.01). (c) Confocal image of DCs expressing GFP 3 h after transfection with mRNA lipoplexes. (d) Similar to panel c, with the difference that 50% of the mGFP was labeled with Cy5 (red) prior to lipoplex preparation. 2946

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antigen processing and presentation to activate the adaptive immune system.39 DCs express a large array of phagocytic receptors (such as lectin and scavenger receptors) and pathogen receptors that induce phagocytosis of bacteria and large solid particles after binding/attachment to these receptors.40 By contrast, macropinocytosis is not receptor mediated, but characterized by the formation of membrane ruffles to engulf large volumes of fluid into macropinosomes, uncoated vacuoles of 0.2−10 μm.41 It is characterized by the actin-dependent reorganization of the plasma membrane, leading to the formation of so-called macropinosomes.24 It occurs in many cell types in the context of nonselective uptake of macromolecules and pathogens.41 In antigen presenting cells macropinocytosis and receptor mediated phagocytosis are exploited by many viruses and bacteria to gain access into the cells, and both pathways contribute to antigen presentation.37,40 The question to be answered is why macropinocytosis would be an advantageous route for transfection mediated by mRNA− Lipofectamine complexes. It is generally accepted that, after having been taken up, lipoplexes are located in endosomes. To ensure transfection they should timely escape from this compartment. According to the model proposed by Xu and Szoka,11 formation of neutral pairs of lipoplex-derived cationic lipids and negatively charged phospholipids present in the endosomal membrane leads to release of the nucleic acid into the cytosol. Obviously, rapid maturation of early endosomes into late endosomes, that later fuse with lysosomes, leaves a very narrow time window for intact mRNA to be released to the cytosol. Therefore, endocytosis pathways avoiding the lysosomal compartment would be beneficial for nucleic acid delivery. Indeed it has been demonstrated by several groups.13,42,43 Moreover, increased levels of transfection were achieved by blocking trafficking of lipo- and polyplexes to the lysosomal compartment.44 Interestingly, very recent results published by Le Roux and colleagues45 elegantly demonstrate that MoDCs have a unique capacity to store macropinocytosed antigens in late endosomes without leading to their degradation. Moreover, these antigens subsequently could be exocytosed in their native form and were able to activate B cells. Since mRNA is quite stable in mild acidic pH (4−5), it is conceivable that it is not denatured under the acidic conditions typical for the lysosomal compartment. It is likely, however, that it is degraded by acid ribonucleases present in this organelle.46 A drop in the luminal pH might additionally contribute to weakening of the interaction between mRNA and a cationic lipid, which normally provides protection against enzymatic degradation. Last but not least also the lipidic part of the complexes might be degraded by (phospho)lipases present in the lysosomal compartment, which might interfere with the mechanism of mRNA release proposed by Xu and Szoka.11 Compared to plasmid DNA (pDNA) mediated gene transfer, mRNA is much more efficient in transfecting nondividing cells. This was recently shown by Andries and colleagues, who compared transfection efficiencies of lipoplexes carrying mRNA or pDNA in respiratory cells.47 The authors reported that it takes more time for pDNA to be expressed, which is not surprising since pDNA needs to cross the nuclear barrier before transcription and later translation can occur. Furthermore, it has been demonstrated that transfection with mRNA lipoplexes was independent of the cell division. The fraction of GFPpositive cells was higher for cells transfected with mRNA than pDNA. However, it was observed that 24 h after transfection the GFP fluorescence intensity was lower for mRNA

Figure 3. Genistein and dynamin, both inhibitors of dynaminrelated processes, reduced GFP production by more than 70%. Amiloride, an inhibitor of macropinocytosis, reduced GFP expression by 40%. Treatment with methyl-β-cyclodextrin and methyl palmitate reduced the number of GFP-positive cells but had no effect on mean fluorescence intensities in GFP-positive cells. Chlorpromazine, an inhibitor of clathrin-mediated endocytosis, had no significant effect on transfection mediated by mRNA lipoplexes.



DISCUSSION We tested the effect of a number of chemical inhibitors blocking different endocytosis pathways on the uptake of Lipofectamine−mRNA complexes by immature monocytederived dendritic cells. Since colocalization studies employing a lysosomal marker clearly demonstrated that around 30% of internalized complexes ended up in the lysosomal compartment, we further investigated the impact of these compounds on transfection mediated by the lipoplexes. Combining the results obtained from the uptake and transfection experiments we can draw the following conclusions. First, methyl-β-cyclodextrin, which removes cholesterol from the plasma membranes, strongly reduced the percentage of transfected cells. Also dynasore, blocking dynamin, strongly affected protein expression. Since most types of endocytosis are dynamin- and cholesterol-dependent, these observations confirmed that mRNA lipoplexes are taken up by endocytosis. Second, transfection of MoDCs was unaffected by chlorpromazine. This is fully compatible with the lack of effect of this compound on the uptake of LF− mRNA complexes and demonstrates that clathrin-mediated endocytosis is not involved in the uptake of these lipoplexes. This was to be expected in view of the average lipoplex size of 800 nm. Treatment of the cells with genistein, which interferes with caveolae-mediated endocytosis and macropinocytosis, reduced protein expression by more than 60%. Moreover, transfection mediated by mRNA lipoplexes was very strongly inhibited by cytochalasin D. This inhibitor diminished the uptake of the complexes by more than 60%. These observations strongly point toward phagocytosis and/or macropinocytosis as important mechanism(s) for the internalization of lipoplexes by DCs. It was unexpected, therefore, to observe that amiloride, known to block macropinocytosis, showed only a marginal effect on lipoplex uptake and a very limited effect on overall transfection efficiency. However, it has been found that the mechanisms involved in macropinocytosis in DC significantly differ from those in all other cells examined. It has been reported that amiloride and its analogues do not specifically inhibit macropinocytosis in DC,37,38 which could explain our observations. It should be added that in our preliminary studies (not shown) rottlerin, another macropinocytosis inhibitor,36 induced a strong reduction in protein expression (data not shown). However, the cell viability was strongly affected by this compound at any concentration used (Table 1). For this reason we did not include this observation. Finally, methyl palmitate, shown to inhibit phagocytosis in Kupffer cells, reduced mRNA translation only moderately. Taken together, these findings point to macropinocytosis as the major mechanism involved in the uptake of mRNA− Lipofectamine complexes, that leads to mRNA translation. Our results are in agreement with findings demonstrating that immature DCs preferentially employ macropinocytosis and phagocytosis to sample their environment for subsequent 2947

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Molecular Pharmaceutics

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transfected cells compared to those treated with pDNA. This is due to the fact that once pDNA reaches the nucleus, it keeps producing mRNA copies, which is not possible in the case of mRNA-based transfection. A major drawback of performing exclusion studies with chemical inhibitors is that the effects of the inhibitors are not thoroughly characterized and can be cell type-dependent.19,20 A more specific method, although requiring a time demanding optimization process, is the use of siRNA to knock down specific proteins.18,48 It should be kept in mind, however, that the use of siRNA requires an additional transfection step preceding antigen delivery, which may severely affect viability and maturation of DCs.



CONCLUSION The overall aim of our study was to evaluate which endocytosis pathways are involved in the uptake of Lipofectamine−mRNA complexes by monocyte-derived dendritic cells. Moreover, we focused at determining which of these pathways lead to efficient mRNA translation. Our findings indicate that particularly Lipofectamine−mRNA complexes taken up by macropinocytosis are productive in terms of transfection. This is likely due to the fact that the lipoplexes internalized along this pathway do not encounter conditions which might biologically inactivate mRNA. We believe that our findings will contribute to rational design of delivery systems allowing efficient and safe modification of dendritic cells both in vitro and in vivo.



AUTHOR INFORMATION

Corresponding Author

*Institute of Tropical Medicine of Antwerp, Virology Unit, Nationalestraat 155, Antwerp 2000, Belgium. E-mail: [email protected]. Phone: 32 3 247 63 72. Fax: 32 3 247 63 33. Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS This work was supported by grants from IUAP (InterUniversity Attraction Poles, P6/41 of the Belgian government), FWO (Fund for Scientific Research Flanders, Project No. G.0226.10) and SOFI-B (Secondary Research Funding ITM). W.D.H. is a doctoral fellow of the Institute for Science and Technology (IWT), Flanders.



ABBREVIATIONS USED MoDCs, monocyte-derived dendritic cells; mGag, mRNA encoding HIV-1 Gag protein; mGFP, mRNA encoding green fluorescent protein; LF, Lipofectamine 2000



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Molecular Pharmaceutics

Article

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