Article pubs.acs.org/molecularpharmaceutics
Histone H3 Tail Peptides and Poly(ethylenimine) Have Synergistic Effects for Gene Delivery Meghan J. Reilly,† John D. Larsen,† and Millicent O. Sullivan*,† †
Department of Chemical Engineering, University of Delaware, Newark, Delaware 19716, United States S Supporting Information *
ABSTRACT: This goal of this work was to explore histone H3 tail peptides containing transcriptionally activating modifications for their potential as gene delivery materials. We have found that these H3 tail peptides, in combination with the cationic polymer poly(ethylenimine) (PEI), can effectively bind and protect plasmid DNA. The H3/PEI hybrid polyplexes were found to transfect a substantially larger number of CHO-K1 cells in vitro compared to both polyplexes that were formed with only the H3 peptides and those that were formed with only PEI at the same total charge ratio; however, transfection was similarly high for polyplexes both with and without transcriptionally activating modifications. Transfections with the endolysosomal inhibitors chloroquine and bafilomycin A1 indicated that the H3/PEI hybrid polyplexes exhibited slower uptake and a reduced dependence on endocytic pathways that trafficked to the lysosome, indicating a potentially enhanced reliance on caveolar uptake for efficient gene transfer. In addition, whereas PEI polyplexes typically exhibit a cytotoxic effect, the H3/PEI hybrid polyplexes did not compromise cell viability. In total, the current studies provide new evidence for the potential role for histone-based materials as effective gene transfer agents, and support for the importance of subcellular trafficking for nonviral gene delivery. KEYWORDS: nonviral gene delivery, hybrid polyplexes, histone peptides, transfection
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endosomal buffering.13,14 Despite these promising attributes, PEI has significant cytotoxicity and lacks an intrinsic capacity to enter the nucleus, release its DNA payload, and activate transcription.15 In contrast, some of the advantages of viral vectors include their intrinsic abilities to deliver DNA to the nucleus and uncoat, such that DNA can interact with transcription complexes.16 The importance of these activities has been confirmed by the observation that infection is reduced when the uncoating process is inhibited17 or when delivery is attempted in cell lines that lack the specific, concerted mechanisms for a particular capsid.18 Chromosomal DNA is also regulated by packaging, and tightly bound heterochromatin is well-established as a repressive structure for transcription. Recent work has shown that post-translational modification (PTM) patterns on histone tails regulate chromatin structure and activation, via both structural/biochemical effects (e.g., alterations in histone charge or bulkiness) and recognition by/recruitment of specific effector proteins. However, although previous work has explored the use of histone monomers,19−21 histone fragments,22,23 and histone/polymer mixtures24 as delivery agents, the role for PTMs in gene delivery had not been addressed until recently,25 and little effort has been devoted to the
INTRODUCTION A multitude of common human diseases and disorders are genetic in origin. Due to the lack of successful and precisely targeted pharmacologically based cures for many of these pathologies, gene therapy is being extensively studied as a means to provide more effective treatment options. Thus, the field of gene therapy has garnered significant interest over the past few decades as a potential method to revolutionize the treatment of various diseases such as hemophilia, Parkinson’s, and many varieties of cancer.1,2 In recent years, nonviral methods of delivery have received particular attention due to immunogenic and toxic responses associated with viral vectors.3,4 However, intracellular limitations including inefficient DNA delivery to the nucleus are a common cause of ineffective nonviral gene transfer. Currently, the most frequently utilized materials for nonviral DNA delivery are cationic lipids, polymers, peptides, or some combination thereof.5−10 Complexes are formed via electrostatic associations between the cationic molecule and the negatively charged DNA phosphate backbone. Sufficiently compact, cationic complexes (lipoplexes or polyplexes) can nonspecifically bind to anionic proteoglycan moieties on the cell surface, resulting in endocytic uptake of the vehicles. Once internalized, lipo- and polyplexes must avoid and overcome various intracellular obstacles. For example, polyethylenimine (PEI), one of the most effective agents for in vitro gene delivery,11,12 possesses a large fraction of secondary and tertiary amine groups that are believed to strongly bind to and compact DNA and facilitate its escape from acidifying vesicles by © 2012 American Chemical Society
Received: Revised: Accepted: Published: 1031
July 26, 2011 November 30, 2011 January 26, 2012 January 26, 2012 dx.doi.org/10.1021/mp200372s | Mol. Pharmaceutics 2012, 9, 1031−1040
Molecular Pharmaceutics
Article
PNA labeled with AlexaFluor488 or AlexaFluor555 was obtained at >90% purity from Panagene (Daejeon, Korea). The PNA consisted of a maleimide-TCTCTCTC-OOOJTJTJTJT-Lys with the appropriate AlexaFluor covalently attached to the Lys terminus [O = 8-amino-3,6-dioxactanoic acid; J = pseudoisocytosine]. Polyplex Formation. Polyplexes were formed with the gWIZ plasmid and mixtures of PEI and/or the H3 peptide according to a modification of established protocols.11,36 Briefly, PEI and peptide solutions were formulated separately in 20 mM HEPES buffer at a pH of 6. pDNA solutions were formulated at 20 μg/mL in the same buffer. Polycation−pDNA particles were prepared by adding the desired polycation solution dropwise to an equal volume of pDNA while gently vortexing. Polyplex formation was then allowed to progress at room temperature for 10 min. Polyplexes containing both H3 peptide and PEI were formed by a stepwise process.37 First, the peptide was added to the pDNA and the mixed solution was equilibrated for 10 min. Subsequently, the appropriate amount of PEI was added and the H3/PEI−pDNA mixtures were incubated for an additional 10 min to enable stable particles to form. 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. For the H3containing polyplexes, the N:P ratio was calculated similarly, but the N contribution from the H3 peptide was calculated as the sum of the total number of arginines, lysines, and the Nterminus. Dynamic Light Scattering and Zeta Potential Analyses. Polyplex sizes were determined by dynamic light scattering (DLS) on a Brookhaven Instruments (Brookhaven, CT) ZETAPals with the 90Plus addition. DLS experiments were performed with a 658 nm wavelength solid-state laser at an angle of 90° and a temperature of 25 °C. For each sample measurement, the hydrodynamic diameter was determined by an intensity-weighted analysis on the data from 3 runs of 2 min each. Zeta potential measurements were obtained with the same ZETAPals instrument. The zeta potential for each polyplex sample was determined by analysis of the data from 10 runs of 2 min each. Quantification of Peptide in H3K4/PEI−pDNA Polyplexes. H3K4−pDNA polyplexes were formulated as described with 4 μg of pDNA at N:P ratios of 2.5, 5, 6, 7, and 10. H3K4/PEI−pDNA polyplexes were similarly prepared at N:P ratios 5/5, 6/4, and 7/3. Millipore YM-100 centrifugal filters (Billerica, MA) were prerinsed by centrifugation with 500 μL of ddH2O for 15 min at 14000g. Once activated, samples were added to the filters and centrifuged. The filtrate was then transferred into high-performance liquid chromatography (HPLC) vials and analyzed using a reversed-phase HPLC (rp-HPLC) instrument from Shimadzu, Inc. (Columbia, MD) on a Viva C18 (4.2 mm × 50 mm, 5 μm particle diameter) column from Restek (Lancaster, PA). The amount of peptide in the filtrate was determined using a linear gradient where solvent A and solvent B consisted of 0.1% trifluoroacetic acid (TFA) in ddH2O and 0.1% TFA in acetonitrile, respectively. A flow gradient of 5−95% solvent B with a flow rate of 1 mL/min was run over 15 min, and peptide elution was monitored at 210 nm by absorbance. The areas of the H3 elution peaks were calculated in MATLAB via the trapezoid method. Cell Transfection. The Chinese hamster ovary (CHO) and mouse embryonic fibroblast (NIH/3T3) cell lines used in these studies were obtained from American Type Culture Collection
mechanisms by which histone-based delivery vehicles might initiate gene transfer. Histone H3 tails that are trimethylated at the fourth lysine (H3K4Me3) have recently generated significant interest because they are present at high levels at the transcription start sites of essentially all active genes in humans and other eukaryotes.26−28 Previous studies have linked H3K4Me3 to transcriptional activation via its recognition by the HBO1 histone acetyltransferase (HAT)29−31 and the nucleosome remodeling factor (NURF) complex in humans,32,33 whose activities catalyze the loosening of DNA−histone interactions at promoter regions and the recruitment of the transcriptional machinery.34,35 Recent work in our lab has explored the potential for H3 peptide-based activation of synthetic gene delivery complexes.25 We found that polyplexes containing H3 histone tail peptides interacted with HBO1,25 suggesting that these peptides might aid in polyplex unpackaging and transcription by recruiting natural effectors involved in chromatin activation. Furthermore, when H3-based polyplexes were microinjected into cellular nuclei, the trimethylated polyplexes induced fast transcriptional activation,25 consistent with a potential role for the peptides in promoting the improved activity of synthetic gene delivery vehicles. To build upon these results demonstrating rapid transcriptional activation by histone H3 peptides, we asked whether these histone-based peptides were competent for cellular transfection. We hypothesized that polyplexes formed with a known mediator of cellular uptake, such as PEI, as well as the H3 peptides might exhibit both robust cellular accumulation and robust transcription. Accordingly, we formed polyplexes with various combinations of PEI and trimethylated or nontrimethylated H3 tail peptides, and found that H3/PEIbased polyplexes containing a small amount of PEI transfected substantially higher fractions of CHO-K1 and NIH/3T3 cells in vitro compared to polyplexes that were formed with either only the H3 peptides or only PEI at the same total charge ratio. These polyplexes did not compromise cell viability, whereas polyplexes formed with PEI alone were cytotoxic. In addition, transfections performed in the presence of various endolysosomal inhibitors indicated that the H3/PEI-based polyplexes exhibited slower uptake and a reduced dependence on acidifying endocytic pathways. Thus, our work suggests that histone-based peptides not only enhance the rate of gene expression, as previously established, but also improve the efficiency of gene transfer via altered subcellular trafficking/ improved nuclear delivery. These studies provide new evidence for histone-based materials as biocompatible and efficient gene transfer agents.
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MATERIALS AND METHODS Materials. The H3 tail peptide ARTKQTARKSTGGKAPRKQLATKAA-CONH2 (residues 1−25 of the mammalian histone H3 protein) and an identical sequence with a trimethylated lysine at position 4 (H3K4Me3) were purchased at ≥95% purity from Anaspec (Fremont, CA). 25 kDa branched PEI was obtained from Sigma (St. Louis, MO). The gWIZ (5757 bp) mammalian expression vector coding for green fluorescent protein (GFP) was purchased from Genlantis (San Diego, CA), amplified in DH5α Escherichia coli and purified with a QIAGEN Plasmid Mega Kit (QIAGEN Inc., Valencia, CA) in accordance with the manufacturer’s protocols. The gWIZ vector contains 10 sequential copies of the repeated peptide nucleic acid (PNA) binding site (5′-AGAGAGAG-3′). 1032
dx.doi.org/10.1021/mp200372s | Mol. Pharmaceutics 2012, 9, 1031−1040
Molecular Pharmaceutics
Article
sample was calculated. A total of 10,000 cells were analyzed for each sample.
(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; all cell culture chemicals were purchased from Fisher (Pittsburgh, PA). For transfection, cells were seeded into 6-well plates at a density of 15,000 cells/cm2. Immediately prior to transfection, the cells were washed in PBS and covered in 2 mL of Opti-Mem per well. Polyplex solutions containing 10 μg of DNA/well were added dropwise to the cells 20 h postseeding. After a 4 h incubation with the transfection reagents, the cells were supplemented with 2 mL of media and cultured for the remainder of the incubation period. For transfections with lysosomotropic drugs, the transfection procedure was modified slightly. Cells were incubated for 30 min in the presence of 100 μM chloroquine or 200 nM bafilomycin A1 in Opti-Mem prior to the start of transfection. After the 4 h transfection period, the drug solutions were replaced with media. Cells were imaged with a Leica 6000 fluorescence microscope (Wetzler, Germany). GFP expression was quantified on a FACS Caliber Flow Cytometer (San Jose, CA). For cytometry analyses, cells were collected after imaging and prepared for analysis by standard trypsin-mediated collection protocols. Cells were resuspended in PBS, filtered through a 35 μm nylon mesh to remove aggregates, and stored at 4 °C until analysis. Scattering plots were gated for quantification purposes, and a total of 10,000 cells were analyzed for each cell sample. Dead cells were excluded from the analyses of transfection efficiency. Cell Viability. Dead cells were visualized by fluorescence microscopy. 4 μM solutions of ethidium homodimer-1 (EthD1; Anaspec) were formulated in PBS and incubated with cells for 10 min. Subsequently, cells were washed and imaged with a Leica 6000 fluorescent microscope. The percent of viable cells was also quantified via flow cytometry analysis of the same cells used to quantify GFP expression. Live/dead cell gating was based on forward and side scatter patterns characteristic of dead cells, and verified based on overlap with EthD-1 staining. Scatter plots were analyzed for both untransfected cells and cells transfected with identical polyplexes containing an SV-40 Renilla expression vector (Promega, Madison, WI). This Renilla vector served as a non-GFP-expressing control for identifying and accounting for any shifts in the live cell autofluorescent region due to the presence of the polycation. A total of 10,000 cells were analyzed for each cell sample. Cellular Uptake of Polyplexes. pDNA was fluorescently labeled with PNA-AlexaFluor488 prior to polyplex formation. For labeling, PNA was added to a solution of DNA at a weight ratio of 1:20 and incubated overnight at 37 °C.36−40 Subsequently, polyplexes were formed as described and the labeled polyplexes were used to transfect CHO cells. The transfections were halted after 0.5, 1, 2, or 3 h of polyplex exposure, and extracellularly bound polyplexes were removed with a 10 μg/mL heparin wash for 15 min at 37 °C. Polyplex uptake was assessed by flow cytometry. Cells were collected and prepared for analysis as described. Particle uptake was subsequently analyzed in two ways. To calculate the fraction of cells that had internalized polyplexes, scatter plots from untransfected cells were gated for autofluorescence, and the gates were used to determine the percentages of transfected cells that had internalized particles. To calculate the average polyplex concentrations per transfected cell, the mean fluorescence intensity (MFI) per cell of each transfected cell
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RESULTS H3 Peptides Formed Polyplexes of Sizes Relevant to Endocytic Uptake. DLS analyses were performed to determine whether combinations of the histone H3 peptides and PEI would form compact pDNA polyplexes suitable for gene transfer (Figure 1). The H3 peptides and PEI were mixed
Figure 1. Hydrodynamic diameters of polyplexes as determined by DLS. For the H3/PEI hybrid polyplexes shown, N:P = 6/4 implies a total charge ratio of N:P = 10 with N = 6 from H3 and N = 4 from PEI [or ∼90% (w/w) H3K4 and 10% bPEI]. Each data point represents the mean ± standard deviation for a total of three separately prepared and analyzed samples.
at various ratios and used to form polyplexes. An overall charge ratio of 10 was used for these hybrid polyplexes based on nuclease stability studies (not shown) and nuclear microinjection studies of polyplex unpackaging.25 Both the H3− pDNA polyplexes and the H3/PEI−pDNA hybrid polyplexes were appropriately sized to participate in endocytic uptake (
