Polyplexes Traffic through Caveolae to the Golgi and Endoplasmic

Article pubs.acs.org/molecularpharmaceutics

Polyplexes Traffic through Caveolae to the Golgi and Endoplasmic Reticulum en Route to the Nucleus Meghan J. Reilly,† John D. Larsen,† and Millicent O. Sullivan*,†,‡ †

Department of Chemical Engineering and ‡Delaware Biotechnology Institute, University of Delaware, Newark, Delaware 19716, United States ABSTRACT: The cellular machinery involved in the internalization of nonviral gene carriers and their subsequent trafficking to the nucleus directly impacts their therapeutic efficiency. Hence, identifying key endocytic pathways and organelles that contribute to the successful transfer of polyplexes to the nucleus generates new opportunities for improving carrier design. Previously, we showed that histone H3 tail peptides encoding a sequence known to participate in chromatin activation exhibit synergistic gene delivery activity with poly(ethylenimine) (PEI). Polyplexes containing H3 and PEI exhibited a reduced dependence on endocytic pathways that trafficked to lysosomes, and had enhanced sensitivity to an inhibitor associated with retrograde trafficking through the Golgi apparatus. Thus, we sought to determine whether caveolar uptake and transport through the Golgi and/or endoplasmic reticulum (ER) preceded nuclear delivery. By the use of a panel of chemical endocytic inhibitors, we determined that H3 polyplexes utilized caveolar pathways to a greater degree than PEI polyplexes. Caveolae-mediated endocytosis was found to be a productive route for gene expression by the H3/PEI−pDNA polyplexes, consistent with previous studies of polymer-mediated gene delivery. Additionally, the polyplexes substantially colocalized within the ER after only 5 min of incubation, and utilized retrograde Golgi-to-ER pathways at levels similar to pathogens known to traffic by these routes during infection. The results of this study have expanded our understanding of how caveolar polyplexes are trafficked to cell nuclei, and provide new evidence for the role of Golgi−ER pathways in transfection. These findings suggest new design criteria and opportunities to stragetically target nonviral gene delivery vehicles. KEYWORDS: nonviral gene delivery, polyplexes, histone peptides, PEI, endocytosis, intracellular trafficking, endoplasmic reticulum, Golgi

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expression is most efficient following clathrin-mediated uptake.9 Because clathrin vesicles are believed to traffic to lysosomes more rapidly than caveolae, these findings may suggest that slow or nonacidifying pathways are advantageous for gene transfer in many situations. Our recent findings suggest that caveolar uptake may also be involved in transfection by polyplexes comprised of pDNA, a histone H3 sequence involved in nuclear localization and chromatin activation,12−16 and branched poly(ethylenimine) (PEI).17,18 We have found that polyplexes containing ∼90% (w/w) H3 tail peptides and ∼10% PEI produced significantly more robust transfection and lower cytotoxicity than PEI− pDNA polyplexes19. We hypothesized that the improved activities of the hybrid H3/PEI−pDNA polyplexes as compared with PEI−pDNA polyplexes were caused by their altered intracellular trafficking and enhanced nuclear delivery. H3/ PEI−pDNA polyplexes exhibited slower uptake and a reduced

INTRODUCTION Although nonviral delivery of plasmid DNA (pDNA) promises advantages such as a lack of innate immunogenicity, it faces many complex obstacles that often result in low transfection efficiency. Improved control over the subcellular trafficking of polyplexes has been widely identified as a key hurdle toward improving their activity in the in vivo setting, where gene transfer inefficiencies cannot be easily offset by increasing the concentration of the delivered vehicle.1−4 Endocytosis is the primary mechanism by which nonviral vehicles, including polyplexes, enter cells,5−7 and polyplexes can be internalized by multiple pathways such as clathrin-mediated uptake, caveolar uptake, and macropinocytosis.2−4 It has become increasingly apparent that the endocytic uptake route is a fundamental determinant of intracellular trafficking and activity.1−4 For example, three recent studies have found that the caveolae/ lipid-raft pathway is a more efficient gene delivery pathway than the clathrin pathway for nontargeted branched PEI (bPEI) polyplexes in HeLa cells.8−10 Other studies have also identified the caveolae/lipid-raft pathway as the most efficient gene delivery route for several different polymers and multiple cell types,1,11 although for a few polymer/cell combinations, gene © 2012 American Chemical Society

Received: Revised: Accepted: Published: 1280

November 17, 2011 March 6, 2012 March 15, 2012 March 15, 2012 dx.doi.org/10.1021/mp200583d | Mol. Pharmaceutics 2012, 9, 1280−1290

Molecular Pharmaceutics

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within 5−30 min of incubation, indicating that some direct transport of polyplexes to the ER from the plasma membrane occurred. In addition, the H3/PEI−pDNA polyplexes accumulated significantly in the Golgi and ER over a longer time scale (2−3 h post-transfection), suggesting that retrograde Golgi-toER pathways were also active for these hybrid polyplexes. To determine whether H3/PEI−pDNA polyplex accumulation in the Golgi and ER was linked to caveolar uptake and transfection, we examined the impacts on Golgi and ER accumulation of caveolin-1 and clathrin inhibitors. Inhibitors of caveolin-1 substantially reduced Golgi and ER accumulation, whereas inhibitors of clathrin had a minimal impact. These data indicated that caveolae were the likely source of Golgi- and ERlocalized polyplexes. In addition, because the H3/PEI−pDNA polyplexes exhibited a greater reliance on retrograde Golgi-toER pathways than the PEI−pDNA polyplexes, and also exhibited more efficient transfection, the data suggested that the Golgi and ER may be desirable destinations for polyplexes en route to the nucleus. Collectively, these studies provide the first demonstration of polyplex accumulation in the Golgi and ER during gene delivery, and lend new insight on efficient intracellular trafficking mechanisms by polyplexes that will guide future vehicle design.

dependence on endocytic pathways that trafficked to the lysosome in comparison with PEI−pDNA polyplexes19. Furthermore, treatment with the macrolide antibiotic bafilomycin A1 significantly reduced H3/PEI−pDNA transfection, but had less substantial effects on PEI−pDNA transfection19. The V-type ATPase binding partners of bafilomycin A1 colocalize with caveolin-1,20 and bafilomycin A1 alters retrograde trafficking from the Golgi complex.21 Thus, these results may suggest that trafficking through caveolae and the Golgi complex represents a productive pathway that enhances H3/PEI−pDNA polyplex delivery to the nucleus. The initial goal of this work was to determine the endocytic machinery involved in H3/PEI−pDNA uptake and transfection. Various chemical inhibitors of the endocytic machinery were utilized to gain understanding of polyplex endocytosis and fate in Chinese hamster ovary (CHO) cells. The H3/PEI− pDNA hybrid polyplexes entered cells primarily through endocytosis, as expected,2,4 and cellular internalization was found to be both actin- and dynamin-dependent. These data, as well as data both from immunocytochemical (ICC) colocalization experiments and from uptake experiments performed in the presence of inhibitors of clathrin-dependent uptake or caveolar uptake suggested that uptake occurred primarily via clathrin- or caveolae/lipid-raft-based pathways.22 Pathways dependent on caveolin-1 were further verified as the primary uptake routes that produced gene expression, as transfection was significantly reduced by caveolar inhibitors. In contrast, despite the reductions in uptake in the presence of clathrinmediated inhibitors, clathrin inhibitors increased gene expression by the H3/PEI−pDNA polyplexes, indicating that clathrin-based endocytosis did not produce efficient gene delivery. Based on the collective data obtained, the hybrid H3/ PEI−pDNA polyplexes appeared to possess a much stronger reliance on the caveolar pathway, even in comparison with PEI−pDNA polyplexes, as their performance was more significantly reduced by caveolar inhibitors and enhanced by clathrin inhibitors. Our finding that caveolar uptake was fundamental to successful gene expression by the H3/PEI−pDNA polyplexes mirrored the important role for caveolae in transfection by other polyplexes, and sparked interest in identifying whether there was a role in gene delivery for retrograde polyplex trafficking through the Golgi apparatus and/or endoplasmic reticulum (ER). No previous studies have elucidated the Golgi and ER as intermediates in polyplex transport,23 yet studies focused on caveolin-1-mediated endocytosis have shown that these compartments are important destinations for transport of other cargo. For example, caveolae have been shown to traffic to the ER by several routes, including from intermediate caveosomes,24,25 through retrograde transport from the Golgi,26−28 or directly from the plasma membrane.29,30 In addition, although clathrin vesicles predominantly traffic through endolysosomal compartments,10,31−33 a few cases have been documented in which clathrin-mediated uptake leads to the Golgi/ER. For example, Ming et al. demonstrated that clathrin-mediated endocytosis of oligonucleotide−peptide conjugates led to partial colocalization with the late endosomal GTPase Rab9,34 which mediates trafficking between late endosomes and the trans-Golgi network.35 Ming et al. hypothesized that transport through the trans-Golgi network may represent a pathway that avoids lysosomes. Our data revealed that both the H3/PEI−pDNA polyplexes and PEI−pDNA polyplexes accumulated extensively in the ER

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MATERIALS AND METHODS Materials. H3 tail peptides (ARTKQTARKSTGGKAPRKQLATKAA-CONH2) both with (H3K4Me3) and without (H3K4) the lysine at position 4 trimethylated were purchased from Anaspec (Fremont, CA) at ≥95% purity. The gWIZ (5757 bps) mammalian expression plasmid encoding green fluorescent protein (GFP) was obtained from Genlantis (San Diego, CA), amplified in DH5α Escherichia coli in Lysogeny Broth, and purified with a QIAGEN Plasmid Mega Kit (QIAGEN Inc., Valencia, CA) following the manufacturer’s protocols. Antibodies against clathrin (monoclonal mouse IgG1 anti-clathrin-membrane vesicle marker; ab2731) and caveolin-1 (polyclonal rabbit IgG anti-caveolin-1; ab2910) were obtained from AbCam (Cambridge, MA). Secondary antibodies (AlexaFluor488 anti-mouse IgG and AlexaFluor488 anti-rabbit IgG) and Hoechst dye were obtained from Invitrogen (Carlsbad, CA). CellLight reagents were obtained from Invitrogen. Peptide nucleic acids (PNAs) were custom synthesized and purified to >90% by Panagene (Daejeon, Korea). Cell culture reagents were purchased from Fisher (Pittsburgh, PA). Branched PEI (25 kDa) and all other reagents were purchased at analytical grade from Sigma (St. Louis, MO). Polyplex Formulation. Polyplexes were formed as previously described by condensing pDNA with H3 and then with PEI, or with PEI alone at various N:P ratios in 20 mM 4(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) buffer at a pH of 6.19 The N:P ratio for the PEI−pDNA polyplexes was calculated as the ratio of the number of amines in the polymer to the number of phosphates in the pDNA, whereas with the H3 peptides, the N value was calculated as the sum of the total number of arginines, lysines, and the Nterminus. For mixtures of H3 and PEI, mixed charge ratios were calculated and reported [for example, N:P = 6/4 implies a total N:P ratio of 10, with N = 6 from H3 and N = 4 from PEI, or ∼90% (w/w) H3K4 and 10% bPEI]. For polyplex uptake and colocalization studies, polyplexes were formed with pDNA that was prelabeled with PNA-AlexaFluor488 or PNA-AlexaFluor555 at a ratio of 1:20 (DNA:PNA), as previously described.19,36 1281

dx.doi.org/10.1021/mp200583d | Mol. Pharmaceutics 2012, 9, 1280−1290


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Table 1. Inhibitors Utilized in Endocytic Uptake and Trafficking Studies chemical

abbreviation

inhibition target

concentration

preincubation time (min)

dynasore cytochalasin D chlorpromazine filipin complex III genistein

Dyn CytD CPZ Fil Gen

all dynamin dependent uptake all actin-dependent pathways clathrin-dependent uptake lipid-raft-dependent uptake caveolin-dependent uptake

80 μM 25 μM 2 μM 1 μg/mL 50 μg/mL

30 15 30 60 30

Cell Culture and Synchronization. CHO-K1 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA). The cells were cultured according to ATCC protocols at 37 °C and 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin−streptomycin. For cell synchronization, CHO cells were plated at ∼7200 cells/cm2. Twenty-four hours after plating, lovastatin solutions in medium were added to cells at a final concentration of 10 μM, and the cells were incubated for 32−36 h.37,38 In order to resume the cell division cycle, the medium containing lovastatin was removed, the cells were washed with phosphate buffered saline (PBS), and fresh medium was added to cells. Inhibitor Treatment. For experiments with endocytic inhibitors (Table 1), cells were treated with the inhibitors prior to as well as during transfection. Preliminary experiments were performed to determine drug concentrations and exposure times that would have a minimal effect on cellular viability, while maintaining the inhibitory effects of the drug (data not shown). Conditions were selected that enabled cells to maintain >90% viability after 24 h of treatment. The general ranges of tested drug concentrations and preincubation times were based on previously reported conditions.1,2,8,33,39−43 Cellular Transfection and Polyplex Uptake Studies. For studies of transfection efficiency, CHO cells were plated into 6-well plates at a density of 15,000 cells/cm2 and cultured in Opti-Mem (Fisher). Immediately prior to transfection, the cells were washed in PBS and covered in 2 mL of Opti-Mem or Opti-Mem containing an endocytic inhibitor. 250 μL of polyplex solution containing 5 μg of DNA was added dropwise to its respective well at 20 h post-seeding and incubated with the cells for 2 h. Subsequently, wells were supplemented with medium or with medium containing the appropriate endocytic inhibitor. Analyses of GFP gene expression were conducted 24 h after transfection. Cells were imaged with a Leica 6000 fluorescence microscope (Wetzler, Germany), and expression was quantified on the FACS Caliber Flow Cytometer. For cytometry analyses, cells were collected and prepared using the same methods as for uptake quantification. Scattering plots were gated for quantification purposes, and a total of 10,000 cells were analyzed for each cell sample. For polyplex uptake studies, transfections with AlexaFluor488-labeled polyplexes were performed as described and ended after a 2 h exposure to polyplexes. Extracellularly bound polyplexes were removed with a 10 μg/mL heparin wash for 15 min at 37 °C, and uptake was subsequently quantified on a FACS Caliber Flow Cytometer (San Jose, CA). Cells were collected, prepared, and analyzed by FACS analysis as described. Immunocytochemistry and Cell Staining. All CHO cells for ICC imaging experiments were plated in 8-well glass bottom plates from Lab Tek (Thermo Fisher Scientific, Waltham, MA) and synchronized as described above. Cells were then incubated for an additional 16 h so that transfection

could take place during the S phase of cell division. Each well was transfected with 50 μL of polyplex suspension containing 1 μg of pDNA labeled with PNA-AlexaFluor555. After a 2 h transfection with polyplexes, cells were rinsed with PBS, washed with 10 μg/mL heparin, washed again with PBS, and fixed with 4% paraformaldehyde in PBS for 15 min. Cells were subsequently permeabilized with 0.2% Tween-20 in PBS (PBSt) and blocked with 3% BSA in 0.2% PBSt. Anti-clathrin and anti-caveolin-1 antibodies were incubated with cells overnight at 4 °C at working dilutions of 1:500 in blocking solutions. Cells were subsequently rinsed three times with PBS and incubated with AlexaFluor488 anti-mouse IgG or AlexaFluor488 anti-rabbit IgG in blocking buffer for 1 h at room temperature. After secondary antibody treatment, cells were rinsed three more times with PBS, cell nuclei were stained with 4 μg/mL Hoechst dye solution, and samples were stored at 4 °C prior to imaging. To determine polyplex colocalization with specific organelles of interest, CellLight Golgi-GFP and ER-GFP were used to stain the Golgi apparatus and ER, respectively. CHO cells were cultured for 24 h, transduced with a given CellLight BacMam baculovirus expression and delivery reagent in medium for 24 h following the manufacturer’s protocol, and stored for future use in liquid N2. BacMam transduction stimulates cellular synthesis of GFP fusion proteins with key intracellular enzymes, including the Golgi-resident enzyme N-acetylgalactosaminyltransferase 2 and an ER signal sequence composed of calreticulin and KDEL. For the colocalization studies, Golgior ER-labeled cells were plated in 8-well glass bottom plates and transfected as described with 35 μL of the polyplex suspension containing 0.7 μg of pDNA labeled with PNAAlexaFluor555. Cells were subsequently fixed and stained with Hoechst dye. Confocal Microscopy and Quantification of Polyplex Colocalization. Cell imaging was performed with a 40× water immersion objective (NA 0.55) on an LSM 5 LIVE Duo microscope (Carl Zeiss, Inc., Thornwood, NY) equipped with appropriate lasers and filters for the selected fluorescent dyes. Volocity Imaging Software (PerkinElmer, Waltham, MA) was utilized for image analysis and quantification of colocalization, where the locations of polyplexes and organelles were determined from measurement statistics associated with individual voxel intensities. The fraction of polyplexes (red voxels) that colocalized with the vesicle or organelle of interest (green voxels) was analyzed by calculation of the Manders coefficient (Mr),44 which represents the sum of the colocalized red intensity divided by the sum of the total red intensity. Minimum values for red and green intensities were automatically determined by Volocity for each image, and these minimum values were set as a threshold to distinguish signal from background. 1282



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RESULTS Caveolar Uptake Leads to Gene Expression by Polyplexes. Cells were transfected in the presence of established chemical inhibitors of endocytosis to elucidate the potential pathways involved in H3/PEI−pDNA polyplex uptake and trafficking.39 H3-based polyplexes containing two different H3 variants [trimethylated (H3K4Me3) and unmethylated (H3K4)] were tested to determine whether previously observed differences in uptake rate of these polyplexes were caused by differences in endocytic trafficking.19 Cells were pretreated with the inhibitors at concentrations that preserved cell viability while maintaining inhibitory effects (Table 1) and, subsequently, were transfected with AlexaFluor488-labeled polyplexes in the presence of the inhibitor. Flow cytometry was used to measure the impacts of inhibitor treatment on polyplex uptake and GFP expression (Figure 1). To verify that active uptake pathways were involved in polyplex internal-

ization, the roles of dynamin and actin were explored. Dynamin is a key protein that facilitates the invagination and scission of budding vesicles from the plasma membrane, and is considered critical for both clathrin-mediated and caveolar uptake. Dynasore (Dyn), an inhibitor of dynamin function, reduced polyplex uptake and transfection efficiency almost entirely for both the H3/PEI−pDNA polyplexes and the PEI−pDNA polyplexes (Figure 1), suggesting clathrin-based and caveolar endocytosis as predominant uptake routes.27,42,45 Actin filaments have also been shown to play a role in vesicle budding and cytosolic release of the detached vesicle.46,47 Actin is known to be involved in clathrin-mediated uptake,48 caveolar uptake,49 and macropinocytosis.50 The fungal metabolite, cytochalasin D (CytD), is known to bind the ends of F-actin with high affinity, inducing the depolymerization of actin filaments.39 The disruption of the actin cytoskeleton also substantially reduced uptake and transfection for both the H3/ PEI−pDNA polyplexes and PEI−pDNA polyplexes, implicating actin-dependent uptake as a vital pathway for delivery. To distinguish the role of clathrin vs caveolar pathways, inhibitors targeting these specific endocytic routes were applied. Chlorpromazine (CPZ), an inhibitor of clathrin-dependent uptake that functions by interfering with the assembly of clathrin at the cell surface, was used to disrupt clathrin-coated pit formation.33 Clathrin inhibition by CPZ produced an approximately 50% reduction in polyplex uptake (Figure 1a), both for H3/PEI−pDNA polyplexes and for PEI−pDNA polyplexes, suggesting that clathrin was significantly involved in polyplex internalization. However, despite the role for clathrin in endocytosis, CPZ treatment did not affect transfection by the PEI−pDNA polyplexes and increased transfection by the H3/ PEI−pDNA polyplexes (Figure 1b). This result supports clathrin-mediated endocytosis as a nonefficient pathway for delivery to the nucleus. Two caveolar inhibitors, filipin complex III (Fil) and genistein (Gen), were used to probe the role for caveolar internalization of polyplexes. Fil is a sterol-binding agent that interferes with lipid-raft-mediated uptake,51 and Gen inhibits tyrosine kinase activity, preventing the phosphorylation of caveolin.43 Both are known to interfere with caveolar uptake and trafficking. Gen had a greater impact on uptake, but both inhibitors significantly reduced H3/PEI−pDNA endocytosis. Both Fil and Gen exposure also significantly reduced transfection efficiency for the H3/PEI−pDNA hybrid particles. Interestingly, Fil had no detectable effect on PEI−pDNA polyplex uptake, but did reduce transfection efficiency, whereas Gen reduced PEI−pDNA uptake, but did not significantly reduce transfection efficiency. This result suggests that PEI− pDNA polyplexes may be separately associating and internalizing with lipid-rafts and caveolae, which are similar, but differ in their incorporation of caveolin-1.49 Polyplex Endocytosis Is Mediated Primarily by both Clathrin and Caveolin. The extents of clathrin-mediated and caveolar endocytosis were further assessed by utilizing fluorescence microscopy to determine whether fluorescently labeled polyplexes colocalized with clathrin or caveolin-1 (cav1), which were visualized via immunostaining (Figure 2a−f). The amount of colocalization with each trafficking protein was quantified by a Manders coefficient (Mr)44 analysis of images obtained on a confocal microscope (Figure 2g). The Manders coefficient is a widely used parameter describing the proportion of coincidence between one color signal and another in a confocal fluorescence image, and is a dimensionless measure of the true degree of colocalization.52 These data corroborated the

Figure 1. In vitro transfection in the presence of endocytic inhibitors. H3K4/PEI, N:P = 6/4 (black); H3K4Me3/PEI, N:P = 6/4 (white); PEI, N:P = 10 (gray). (a) Cellular uptake efficiencies of fluorescently labeled pDNA polyplexes at 2 h post-transfection. (b) Cell transfection at 24 h post-transfection. Data were obtained with flow cytometry measurements, and results are presented as the normalized values relative to a respective sample control (−), where no inhibitors were added. Each data point represents the mean ± standard error for a total at least three separately prepared and analyzed samples. * indicates a statistically significant difference relative to the respective polyplex control (p < 0.05). 1283



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Figure 2. Colocalization (yellow) of fluorescently labeled pDNA polyplexes (red) with clathrin (a−c) or cav-1 (d−f) (green). (a−f) Representative confocal microscopy z-stack images of cells with nuclei stained with Hoechst (blue) following 2 h transfection with the indicated polyplexes. The scale bar [shown in (a)] = 10 μm. (g) Quantification of colocalization from confocal microscopy images was performed with Volocity Image Analysis software. Mr values range from 0 (no colocalization) to 1 (complete colocalization of red voxels with green voxels). H3K4/PEI, N:P = 6/4 (black); H3K4Me3/PEI, N:P = 6/4 (white); PEI, N:P = 10 (gray). Each data point represents the mean ± standard error for a total of 20 to 50 cells. * indicates a statistically significant difference among polyplexes relative to clathrin or cav-1 (p < 0.05).

H3/PEI−pDNA hybrid polyplexes (Figure 3b). Collectively, these findings suggested that the inhibitory effects of CPZ, Fil, and Gen exhibited the expected target specificity. Polyplexes Traffic through the ER and Golgi. Previous work has demonstrated that different organelles can be targeted by a given endocytic pathway, leading to divergent intracellular trafficking of a specific type of cargo. For example, Le and Nabi have reported that different types of cargo, each similarly internalized by caveolae-mediated endocytosis, experienced distinct trafficking to different organelles such as the Golgi apparatus or the ER.29 The trans-Golgi network has been suggested as a potential route to avoid lysosomes en route to the nucleus.34 Thus, because our trafficking studies indicated that caveolar uptake led to efficient gene expression by H3/ PEI−pDNA polyplexes, we asked whether the Golgi and/or ER might be intermediate destinations of these caveolar polyplexes. Polyplex colocalization with markers of the Golgi and the ER was quantified from images obtained by confocal microscopy (Figure 4). Colocalization with both the Golgi (N-acetylgalactosaminyltransferase 2) and ER (signal sequence of calreticulin and KDEL) was observed for the H3/PEI−pDNA polyplexes, with rapid and substantial accumulation of these polyplexes in the ER within 5 min, and a gradual increase in ER accumulation over 30 min. Golgi accumulation by the H3/ PEI−pDNA polyplexes occurred at a lower level and was

involvement of both clathrin- and caveolae-mediated transport. PEI−pDNA polyplexes were internalized by clathrin vesicles much more readily than by caveolae, but overall, clathrin- and caveolin-associated uptake only accounted for ∼50% of the total PEI-based polyplexes, consistent with previous reports suggesting that macropinocytosis is a significant mechanism for PEI-mediated polyplex internalization.53 Interestingly, the H3/ PEI−pDNA polyplexes colocalized with both clathrin and cav-1 to a substantially greater extent than the PEI−pDNA polyplexes, suggesting that these internalization pathways were dominant for the H3-based polyplexes. Control immunostaining experiments in which transfections and staining were performed in the absence of either primary antibodies or secondary antibodies ensured that fluorescent staining for clathrin or caveolin-1 was specific to the targeted proteins (data not shown). The impacts of CPZ, Fil, and Gen disruption on clathrinmediated and caveolar polyplex uptake were also investigated through immunostaining. CPZ reduced colocalization with clathrin by 40−60% for all types of polyplexes, whereas Gen did not affect clathrin colocalization with PEI−pDNA polyplexes and increased clathrin colocalization for the H3/PEI−pDNA polyplexes (Figure 3a). Immunostaining for cav-1 revealed no change in colocalization due to CPZ for any of the samples, but a decrease in colocalization due to the presence of Gen for the 1284



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consistent with previously published work implicating caveolin1-dependent retrograde trafficking to these compartments.29 Collectively, the inhibitor studies indicated that while both clathrin-mediated and caveolar endocytosis contributed to retrograde trafficking by polyplexes, trafficking through the Golgi/ER was the primary route utilized by polyplex-containing caveolae.

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DISCUSSION Histone H3-containing polyplexes were developed with the intention of creating materials that would facilitate nuclear delivery and transcriptional activation of pDNA within the nucleus. Our prior studies demonstrated that H3-based polyplexes accumulated in the nucleus after delivery to the cytosol54, and that H3 peptides had synergistic activities with PEI that led to improved gene delivery. Specifically, combining H3K4 or H3K4Me3 with PEI significantly enhanced cell transfection without compromising viability, as compared with either H3- or PEI-based transfection19. Thus, we hypothesized that improved nuclear delivery might underlie the strong activity of the hybrid polyplexes, and we sought to identify key reasons underlying the synergies between the H3 peptides and PEI. Previously, we observed that H3/PEI−pDNA polyplexes (particularly those formed with H3K4Me3) were more gradually internalized by cells and were more substantially impacted by inhibitors associated with caveolar trafficking than PEI polyplexes19. This led us to postulate that caveolaemediated endocytosis was playing a major role in H3/PEI− pDNA polyplex trafficking since caveolar uptake kinetics are slower than those of clathrin.55,56 Since conventional caveolar trafficking involves both dynamin- and actin-mediated internalization, whereas macropinocytosis is actin-dependent but is not believed to be dependent on dynamin,57 the relevance of active endocytic uptake was explored with the chemical inhibitors Dyn and CytD.29 Both Dyn and CytD almost entirely negated uptake and expression, highlighting the important roles of both dynamin and actin in polyplex internalization and gene delivery, and confirming that active endocytic uptake took place. This led us to focus on the clathrin and caveolar pathways, which rely on both actin48,49 and dynamin,58,59 and are known to participate in the endocytosis of PEI.8 Caveolar trafficking also involves cholesterol sensitivity and tyrosine kinase activation by cav-1, whereas clathrin-based trafficking involves clathrin recruitment and assembly by AP-2 binding to the plasma membrane. Thus, we sought to elucidate the specific roles for caveolae vs clathrin vesicles by analyzing the effects on uptake and expression of exposing cells to specific clathrin or caveolar pathway inhibitors prior to transfection. Although all the polyplexes in this study were shown to participate in both clathrin and caveolar endocytosis (Figure 2), we confirmed that the caveolar uptake pathway was utilized by approximately 3-fold more of the H3/PEI−pDNA polyplexes as compared with the PEI−pDNA polyplexes. The caveolar pathway was essential for transfection by H3/PEI−pDNA polyplexes. Inhibiting caveolar uptake had a negative impact on the resulting gene expression, whereas inhibiting clathrin uptake actually enhanced expression, even though pharmacological obstruction of either pathway reduced polyplex uptake (Figure 1). An enhancement in expression by clathrin inhibition has previously been observed with other types of polyplexes,1 and led us to infer that H3/PEI−pDNA polyplexes participating in clathrin-mediated endocytosis were not

Figure 3. The effect of endocytic inhibitors on polyplex colocalization with clathrin (a) or cav-1 (b) at 2 h post-transfection. Quantification of colocalization from confocal microscopy images was performed with Volocity Image Analysis software. H3K4/PEI, N:P = 6/4 (black); H3K4Me3/PEI, N:P = 6/4 (white); PEI, N:P = 10 (gray). Data were normalized relative to the respective sample control (−), where no inhibitors were added. Each data point represents the mean ± standard error for a total of 20 to 50 cells. * indicates a statistically significant difference relative to the respective polyplex control (p < 0.05).

slower, with increasing accumulation over at least a 3 h period following transfection. The PEI−pDNA polyplexes also accumulated rapidly in the ER for the first 15−30 min following transfection, and then accumulation began to decrease. PEI−pDNA accumulation occurred at a low level in the Golgi, suggesting that trafficking to the ER, but not the Golgi, might play a role in nuclear delivery and gene expression by these purely PEI-based polyplexes. Caveolar Uptake of Polyplexes Leads to the Golgi and ER. To determine directly whether clathrin-mediated or caveolar uptake led to subsequent polyplex accumulation in the Golgi or ER, polyplex−Golgi and polyplex−ER colocalization were assessed after cell treatment with either CPZ or Gen (Figure 5). CPZ had little effect on accumulation in the Golgi, but reduced accumulation in the ER for all polyplexes. In contrast, Gen treatment reduced accumulation in the Golgi by ∼65% for the trimethylated H3/PEI−pDNA polyplexes, and reduced ER accumulation by ∼60−80% for all polyplexes. This finding indicated that a portion of the trimethylated H3-based polyplexes were shuttled to the Golgi, and a substantial fraction were shuttled to the ER as a result of tyrosine kinase activation, 1285



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Figure 4. Colocalization (yellow) of fluorescently labeled pDNA polyplexes (red) with the Golgi (a−c) or ER (d−f) (green). (a−f) Representative confocal microscopy z-stack images of cells with nuclei stained with Hoechst (blue) following a 2 h transfection with the indicated polyplexes. The scale bar [shown in (a)] = 10 μm. (g−h) Quantification of colocalization with the Golgi (g) or ER (h) from confocal microscopy images at different times post-transfection, performed by Volocity Image Analysis software. Mr values range from 0 (no colocalization) to 1 (complete colocalization of red voxels with green voxels). H3K4/PEI, N:P = 6/4 (black); H3K4Me3/PEI, N:P = 6/4 (white); PEI, N:P = 10 (gray). Each data point represents the mean ± standard error for a total of 10 to 25 cells. * indicates a statistically significant difference among polyplexes at a given time point (p < 0.05). # indicates a statistically significant difference from the previous time point for a given polyplex (p < 0.05).

transfection, we believe that a fraction of polyplexes is directly trafficked to the ER. Direct trafficking to the ER has been reported in a few examples, including for caveolae-mediated uptake of autocrine motility factor (AMF).29,30 AMF was shown to accumulate in the ER following only 5 min of incubation, and uptake was negatively impacted by tyrosine kinase inhibition. Since the polyplexes in this study could be detected at significant levels after just 5 min and were also shown to be Gen-sensitive, this strongly supports the assertion that these polyplexes are directly trafficked to the ER (Figure 6). Other cargo, including cholera toxin (CTX) and Simian virus 40 (SV40), target to the ER via slower, retrograde pathways. CTX targets to the ER from the Golgi by a pathway that is sensitive to Fil exposure.26,27 SV40 has been shown to follow a similar, though not identical, route as CTX. SV40 reaches the ER through an intermediate organelle known as the caveosome, via a pathway sensitive to tyrosine kinase activation.24,25 These pathways are also potential contributors to H3/PEI−pDNA delivery. The caveolar inhibitor Gen reduced both transfection and H3-based polyplex colocalization with the Golgi and ER, indicating that retrograde polyplex transit via a Golgi-to-ER route might lead to productive delivery (Figure 6). Although

trafficked to the nucleus, but were instead lost to cytosolic or lysosomal degradation. In contrast, since multiple inhibitors of conventional caveolar trafficking (Dyn, CytD, Fil, Gen) negatively influenced H3/PEI−pDNA uptake and subsequent gene expression, we deemed that caveolae were the primary endocytic contributors to efficient transfection. This finding is in accordance with previous work in which PEI−pDNA polyplexes and other types of polyplexes were observed to transfect cells more efficiently via caveolae, whereas clathrinmediated endocytosis was found to result in lysosomal degradation.8,10 Previous work has revealed that different materials that similarly enter cells via caveolar uptake can experience dissimilar trafficking to numerous organelles once internalized.26,29 To determine the role of key organelles in trafficking, polyplex accumulation in the Golgi and ER were appraised. These organelles were specifically investigated since the literature has revealed that there are distinct caveolar pathways that can target either or both of these organelles, and also because retrograde trafficking via the trans-Golgi network has previously been cited as a potential route by which nucleic acids might avoid lysosomes.34 Since the polyplexes could be detected in the ER at significant levels within 5 min of 1286



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Overall, few differences were observed in the trafficking and gene expression of the H3K4/PEI−pDNA polyplexes and the H3K4Me3/PEI−pDNA polyplexes, consistent with previously observed minor differences in uptake rates of these polyplexes. Although slightly more of the polyplexes formed with the trimethylated H3 peptide colocalized in the Golgi by 3 h (Figure 4g), and the trimethylated polyplexes were somewhat more sensitive to Gen exposure, the trafficking routes and activities of the H3-based polyplexes were generally very similar.

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CONCLUSIONS A more evolved understanding of the endocytic and subcellular machinery contributing to polyplex trafficking from the plasma membrane to the nucleus is a necessary step toward the creation of efficient nonviral vehicles. Throughout this work, various published studies have been cited that deal with the identification of optimal uptake pathways using different cell lines and vehicles; however, a comprehensive understanding of how polyplexes travel from internalization vesicles to the nucleus is still lacking, even for conventional gene carriers like PEI.23 The studies presented in the described work provide new information on how organelles in the perinuclear region specifically the Golgi apparatus and the ERcan influence polyplex fate. The data presented in this work explore the importance of subcellular trafficking pathways on observed synergies in activity between biomimetic histone H3 tail peptides and PEI. Though multiple pathways played a role in polyplex transport, we ultimately found that a significantly greater fraction of H3/PEI−pDNA polyplexes utilized endocytic pathways involving either caveolae or clathrin as compared with PEI−pDNA polyplexes, which are also known to utilize macropinocytosis. However, while both caveolar and clathrinmediated uptake contributed to H3/PEI−pDNA polyplex internalization, caveolar uptake appeared to be the predominant pathway leading to gene expression, consistent with several previous studies identifying caveolar uptake as a more productive endocytic route. These findings suggested that one reason for the observed enhancement in H3/PEI−pDNA activity over PEI−pDNA activity was the enhanced utilization of caveolar pathways by the H3/PEI−pDNA polyplexes. A new discovery in the current work was the identification of the Golgi and ER as key intermediates for caveolar polyplexes en route to the nucleus. We found that once internalized, H3 polyplexes utilized both direct trafficking to the ER and retrograde Golgi-to-ER transport at levels similar to pathogens such as SV40. Although we cannot rule out other destinations for caveolar cargo, since not all caveolar polyplexes were accounted for in the Golgi/ER, a substantial fraction of initially caveolar H3 polyplexes trafficked to the Golgi and/or ER, and caveolar inhibitors almost entirely reduced Golgi/ER colocalization. In contrast, clathrin vesicles did not substantially lead to the Golgi or ER, and may instead traffic to various nonproductive destinations such as lysosomes or the cytosol. Trafficking through retrograde Golgi−ER pathways also represented a key difference between the behaviors of H3/ PEI−pDNA polyplexes and PEI−pDNA polyplexes. Thus, the reduced activities of PEI polyplexes may be related both to their reduced dependence on caveolae and to their lack of utilization of retrograde pathways. These retrograde pathways may enable polyplexes to avoid lysosomes en route to the nucleus and prior to gene expression. Our current experiments are focused on

Figure 5. The effect of endocytic inhibitors on polyplex colocalization with the Golgi (a) or the ER (b) at 2 h post-transfection. Quantification of colocalization from confocal microscopy images was performed with Volocity Image Analysis software. H3K4/PEI, N:P = 6/4 (black); H3K4Me3/PEI, N:P = 6/4 (white); PEI, N:P = 10 (gray). Data were normalized relative to the respective sample control (−), where no inhibitors were added. Each data point represents the mean ± standard error for a total of 15 to 35 cells. * indicates a statistically significant difference relative to the respective polyplex control (p < 0.05).

accumulation of polyplexes within the Golgi was overall reasonably low, the observed levels were similar to levels reported previously for SV40,61 and were consistent with the Golgi as a transient intermediate during polyplex routing and delivery. One surprising result was that CPZ also appeared to reduce the amount of polyplex accumulation in the ER, indicating that clathrin-dependent uptake might also influence trafficking to this organelle. Clathrin-mediated endocytosis has been shown to predominantly traffic polyplexes and other cargo to endosomes and/or lysosomes.62 However, there have been a few observed instances of trafficking to the ER. For example, subtilase cytotoxin (SubAB) undergoes clathrin-dependent accumulation in the ER via retrograde transport from the Golgi.63 Therefore, some observed effect on ER accumulation due to CPZ exposure is not unreasonable. However, since CPZ did not affect polyplex−Golgi colocalization, and had less pronounced effects on polyplex−ER colocalization than Gen, Golgi/ER trafficking via the clathrin pathway was considered less influential on successful delivery than the caveolar route. 1287



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Figure 6. Schematic detailing known and proposed uptake and intracellular trafficking mechanisms for PEI and H3/PEI hybrid polyplexes. (i) Macropinocytosis;1,48,53 (ii) clathrin-mediated endocytosis;1,8−10,17,60 and (iii) caveolae-mediated endocytosis.1,8−10 Arrows in black indicate known pathways for polyplexes that have been identified in previous literature, which are referenced accordingly. Arrows in pink indicate new potential pathways for polyplexes, as suggested by the studies presented in this work.

equipment and software used for all 3D cellular imaging and analysis.

better elucidating these potential retrograde mechanisms by which polyplexes may traffic to the nucleus. Collectively, this study indicates that incorporating direct methods for targeting polyplexes to caveolae and/or the Golgi/ ER might be beneficial for improved nuclear delivery and gene expression. The inclusion of a histone sequence relevant to chromatin unpackaging into H3/PEI hybrid polyplexes led to greater colocalization with caveolin-1 and greater sensitivity to caveolar pathway inhibitors in comparison with PEI polyplexes. The reasons underlying this enhanced utilization of the caveolar pathway are unclear, as caveolae are not major contributors to histone internalization.64−66 However, histones are known to travel within the trans-Golgi network during post-translational modification.67,68 Therefore, histone peptide interactions with trafficking components in the trans-Golgi network may underlie the enhanced utilization of retrograde pathways by H3/PEI− pDNA vehicles. The possible roles for these interactions in directed trafficking are a subject of our further exploration.

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*150 Academy St., Newark, DE 19716. Phone: (302) 8318072. Fax: (302) 831-3009. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This material is based upon work supported by the National Science Foundation under Grant No. DMR-0746458. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Science Foundation. We acknowledge the Center for Translational Cancer Research Core Facility for use of their flow cytometer and the Delaware Biotechnology Institute Bioimaging Facility for use of their confocal microscopes. We thank Dr. Jeffrey L. Caplan for training and continuing guidance with the 1288



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