Using the Epigenetic Code To Promote the Unpackaging and

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Using the Epigenetic Code To Promote the Unpackaging and Transcriptional Activation of DNA Polyplexes for Gene Delivery John D. Larsen,† Meghan J. Reilly,† and Millicent O. Sullivan*,† †

Department of Chemical Engineering, University of Delaware, 150 Academy Street, Newark, Delaware 19716, United States S Supporting Information *

ABSTRACT: Nonviral gene delivery has seen limited clinical application due in part to the inefficiency with which most nonviral vehicles navigate the intracellular gene delivery pathway. One key problem is the inability of most DNA-packaging materials to release DNA and enable its efficient transcription. Thus, our aim was to develop gene delivery polyplexes capable of initiating their own transcription upon arrival in the nucleus. We created nuclease-resistant polyplexes with plasmid DNA (pDNA) and post-translationally modified histone 3 (H3K4Me3) tail peptides known to signal transcriptional activation on chromosomal DNA. When the H3K4Me3−pDNA polyplexes were directly microinjected into the nuclei of NIH/3T3 mouse fibroblasts, protein expression occurred earlier and in a greater fraction of cells than when polyethylenimine−pDNA polyplexes were microinjected. The rate of protein expression initiated by the H3K4Me3−pDNA polyplexes was also significantly accelerated in comparison with the rate initiated by non-trimethylated H3−pDNA polyplexes. These differences in protein expression rates were quantified by the development of a noncompartmentalized cellular kinetics model. These results highlight the importance of polyplex unpackaging as a gene delivery barrier, and demonstrate for the first time that the epigenetic code can be utilized in nonviral gene delivery. KEYWORDS: nonviral gene delivery, HBO1, epigenetics, histone 3 protein

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upon polymer hydrolysis.13 Unpackaging of the DNA in the endosome or cytoplasm, however, is not ideal given that plasmid DNA cannot effectively diffuse in the cytoplasm14,15 and has a cytoplasmic half-life of approximately 60 min.16 Recent studies have shown that total cellular DNA release correlates poorly with transfection efficiency.17 Therefore, Grigsby and Leong recently suggested, “an ideal gene carrier would protect DNA from nucleases and provide unrestricted access to polymerases.”18 To create a nonviral gene delivery vehicle capable of such protection and release capabilities, recent advances in the understanding of nature’s “on/off” mechanisms for controlling DNA transcription suggest novel solutions for the improved design of DNA-packaging biomaterials. Chromosomal DNA is condensed by histone octamers into nucleosomes, which contain transcriptionally repressed and active domains known as heterochromatin and euchromatin, respectively.19−21 Transcriptional activation involves the partial release of the DNA from the histone, and is signaled by the reading and writing of histone tail post-translational modifications.22 For example, trimethylation of the position 4 lysine on the histone 3 protein (H3K4Me3) is strongly associated with the 5′ region of actively transcribing genes in a variety of eukaryotes.23,24 In humans,

INTRODUCTION Over the past two decades, there has been intense research in the field of nonviral gene delivery, with limited success.1 One persistent issue is that typical nonviral formulations do not allow the transcriptional machinery to efficiently access the packaged DNA. These polymer- and lipid-based formulations (“polyplexes” and “lipoplexes,” respectively) form via a spontaneous, entropically driven process in which cationic residues on the carrier self-assemble with negatively charged phosphate anions along the backbone of DNA,2 and if sufficient polycation is added, the hydrophobicity of the partially neutralized DNA becomes so high that it loses its extended conformation and is forced to collapse.3 Although this condensation process imparts particle size control and sterically excludes nucleases, the condensed structures are similar to heterochromatin, whose tight packaging inhibits DNA transcription.4 Inefficient polyplex disassembly has been identified as a minimally studied, yet critical, rate-limiting barrier for gene delivery.5−11 Because the same beneficial characteristics (i.e., a high density of positive charge) that promote tight association of a polycation with DNA during gene transport eventually inhibit the activity of the DNA within the nucleus, the design of effective DNA-packaging biomaterials has presented a persistent challenge. To promote the intracellular unpackaging of DNA, current approaches include sulfide-conjugated polymers that promote complex disassembly within the cytoplasm12 and “charge-shifting” polymers that release DNA © 2012 American Chemical Society

Received: Revised: Accepted: Published: 1041

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Peptide Synthesis. The H3 peptide sequence incorporates residues 1−28 of the N-terminal tail of the yeast H3 protein as well as a C-terminal cysteine residue as a potential reactive handle (ARTKQTARKSTGGKAPRKQLASKAARKSGCCONH2, where the italicized residues are exogenous residues added as a putative reactive handle) and was synthesized by solid phase peptide synthesis with a Protein Technologies, Inc. (Tucson, AZ) Tribute series peptide synthesizer and a Tentagel solid phase resin. Cleavage of the peptide from the resin was performed using a cocktail consisting of 5 wt % phenol in 95 vol % trifluoroacetic acid (TFA), 2.5 vol % H2O, and 2.5 vol % triisopropylsilane. Purification of the peptide was performed by reversed-phase high performance liquid chromatography (HPLC), on a UFLC 20 series instrument from Shimadzu, Inc. (Columbia, MD) with a gradient of 0.1% TFA in doubly deionized H2O (ddH2O) (A) and 0.1% TFA in acetonitrile (B) as the mobile phase. The gradient was allowed to run from 10% B to 35% B over 50 min at a flow rate of 5 mL/min through a Viva C18 (21 mm × 150 mm, 5 μm particle diameter) column from Restek (Lancaster, PA). Peptide elution was monitored by absorbance measurements at 210 nm. The [M + 2H]2+ was determined with electrospray ionization mass spectrometry (ESI-MS) on a Thermo Finnigan LCQ MS and was found to be 1,572.6. The experimental molecular weight was found to be 3,143.2 Da (predicted at 3,143.7 Da). This sequence differs minimally from the mammalian analogue, with a single substitution at residue 22 (S/T). Analogous biotinylated peptides were synthesized on a rink amide ChemMatrix resin, with the addition of a GK(biotin) at the C-terminus. These peptides were purified as above, and their molecular weights were determined through matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) MS on a Bruker Daltonics Omniflex System (Billerica, MA) as 3,555 Da and 3,597 Da for the non-trimethylated and the trimethylated H3 peptides, respectively (predicted at 3,553.9 Da and 3,595.9 Da). Biotinylation of PEI. To synthesize the biotinylated-PEI, PEI was dissolved in 10 mL of 0.1 M NaPO4 buffer, pH = 8.0, and the resulting solution was cooled on ice for 15 min. EZ-link sulfo-NHS-LC-biotin was dissolved at a concentration of 1 mg/ mL in 0.1 M NaPO4 buffer, pH = 8.0. Immediately after dissolution, 42 μL of the biotin linker solution was added to 1 mL of the PEI solution, and the resulting mixture was stirred and reacted for 4 h at 4 °C. Subsequently, excess biotin was removed by centrifugal filtration through an Amicon Ultra 10 kDa molecular weight cut off (MWCO) centrifugal filter and the biotinylated-PEI was recovered. The degree of biotinylation was determined using a HABA assay, following the manufacturer’s protocol. The degree of biotinylation was determined to be 1.8 ± 0.4 biotin molecules per PEI molecule. Polyplex Formation and Characterization. DNA polyplexes were formed in 20 mM 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid (HEPES) buffer, pH = 6.0, at a concentration of 20 μg of DNA/mL, according to previously established protocols.35,36 Briefly, solutions of PEI or H3 peptide were added dropwise to the DNA solutions while vortexing. The N:P ratios were determined for PEI as the ratio of the number of PEI amines to the number of DNA phosphates. The N:P ratios were determined for the H3 polyplexes as the ratio of the sum of the number of arginines, lysines, and N-terminal amines to the number of DNA phosphates. The polyplexes were analyzed by agarose gel electrophoresis according to standard protocols. For gel electrophoresis, 1%

H3K4Me3 recruits and binds the HBO1 histone acetyltransferase (HAT),25−27 which catalyzes the acetylation of downstream lysines on the H3 N-terminal tail. Histone acetylation is strongly associated with chromatin activation via its capacity to neutralize and add bulkiness to lysine residues,28,29 which subsequently allows the transcriptional machinery to bind to and transcribe the loosened DNA. H3K4Me3 is also recognized by the NURF (nucleosome remodeling factor) chromatin remodeling complex, which activates chromatin via its capacity to disrupt nucleosomes and recruit transcription factors.30−33 With this knowledge, our aim was to recapitulate on therapeutic DNA the unpackaging and transcriptional activation properties of the trimethylated H3 subunits through the use of the trimethylated H3 N-terminal tails as DNA-packaging biomaterials. We formed polyplexes from plasmid DNA (pDNA) and peptides comprising residues 1−28 of the yeast analogue tail of the H3 protein, as this region is a nuclear localization sequence.34 The H3-based peptides and pDNA formed tightly packaged, nanoscale polyplexes that, prior to their exposure to the intranuclear environment, were inaccessible to proteins like nucleases. When the H3-containing polyplexes were injected into cellular nuclei, the H3-based polyplexes initiated higher levels of transfection than standard, poly(ethylenimine) (PEI)-based polyplexes, and furthermore, polyplexes containing the H3K4Me3 motif initiated gene expression significantly faster than either PEI polyplexes or H3-containing polyplexes that lack the H3K4Me3 motif. These results suggest, for the first time, a role for epigenetically patterned histone fragments in promoting the unpackaging and transcriptional activation of synthetic gene delivery materials.

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MATERIALS AND METHODS Materials. All chemicals were purchased from Sigma (St. Louis, MO), including 25 kDa branched PEI, which was used without further purification. All peptides, except the H3K4Me3 sequence, in which the lysine at the fourth position from the Nterminus was trimethylated, were synthesized using Fmocprotected amino acids obtained from EMD Chemicals (Darmstadt, Germany). Rink amide Tentagel solid phase resin was purchased from Anaspec (Fremont, CA). Rink amide ChemMatrix resin was purchased from PCAS Biomatrix, Inc. (Saint-Jean-sur-Richelieu, Canada). The H3K4Me3 sequence was purchased from Anaspec at >95% purity. Amicon Ultra centrifugal filters were purchased from Millipore (Billerica, MA). 70 kDa dextran conjugated to Texas Red, penicillin, streptomycin, and M-280 streptavidin-coated Dynabeads were obtained from Invitrogen (Eugene, OR). Femtotips II and femtoloaders were purchased from Eppendorf (Hamburg, Germany). An HBO1-enhanced mouse cell lysate was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). An anti-HBO1 rabbit polyclonal antibody was purchased from AbCam (Cambridge, CA), and a donkey anti-rabbit polyclonal antibody conjugated to horseradish peroxidase (HRP) was purchased from GE Healthcare (Piscataway, NJ). The gWIZ plasmid, which encodes for green fluorescent protein (GFP), was purchased from Genlantis (San Diego, CA) and amplified in DH-5α Escherichia coli cells cultured in Lysogeny Broth. The cells were lysed and the amplified plasmid was purified using a Qiagen (Hilden, Germany) MegaPrep kit, in accordance with the manufacturer’s protocol. Dulbecco’s modification of Eagle’s medium (DMEM) and phosphate buffered saline (PBS) solutions were obtained from Mediatech (Manassas, VA). 1042

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Figure 1. Cell-by-cell quantification of GFP intensity for delay time analysis. (a) Representative fluorescent images of a microinjected cell, shown for a cell injected with free pDNA. The indicated times represent minutes postmicroinjection. The scale bar represents 100 μm. (b) Representative data from a sample of cells that were microinjected with free pDNA. The integrated fluorescence intensity in each cell (each line is one cell) was determined through quantitative image analysis. (c) The data shown in (b) were normalized to account for differences in cellular autofluorescence, as well as the standard variability in microinjection volumes from cell to cell.

agarose gels containing 0.5 μg of ethidium bromide/mL were formed in 1× tris/borate/ethylenediaminetetraacetic acid (EDTA) (TBE) buffer. Twenty microliters of each polyplex solution was added to 5 μL of gel loading buffer, and 20 μL of each of these mixtures was added to each well of the gel. The gels were run at 100 V for 1 h and subsequently imaged using a BioRad Gel Doc XR (Hercules, CA). For the nuclease stability assays, 50 μL of each polyplex solution containing 5 mM MgCl2 was incubated with 5 units of DNase I for 2 h at 37 °C. To test the influence of ionic strength on nuclease stability, the polyplexes were incubated either in 20 mM HEPES or in a PBS solution (137 mM NaCl, 0.49 mM MgCl2, 1.5 mM KH2PO4, 2.7 mM KCl, 0.90 mM CaCl2, and 8.1 mM Na2HPO4). The nuclease activity was terminated by adding 8 μL of a buffer composed of 0.16 M EDTA, 0.67 M NaOH, and 0.16 M NaCl, and placing the terminating solutions on ice for 10 min. DNA was subsequently released from the polyplexes by the addition of 2 μL of a 10 mg/mL heparin solution followed by incubation of the heparin−polyplex mixtures for 10 min at 4 °C. The released polyplex solutions were analyzed by gel electrophoresis as previously described. A Brookhaven Instruments (Brookhaven, CT) ZETAPals with the 90Plus addition was used for dynamic light scattering (DLS) analyses. Measurements were performed at ambient temperature with a 658 nm wavelength solid-state laser, and scattering was collected at 90°. Data were analyzed using a second order cumulant fit to obtain an average hydrodynamic diameter of the complexes formed.

Determination of Polyplex Composition. H3−pDNA polyplexes were made with 2 μg of pDNA at N:P ratios of 1.25, 2.5, 5, and 10, as previously described,36−38 and diluted with 90 μL of ddH2O. Amicon Ultra 100 kDa MWCO centrifugal filters were activated by adding 500 μL of ddH2O and centrifuging at 14000g for 15 min. The polyplex samples were subsequently added to the filters and centrifuged at 14000g for 15 min. The filtrate was collected and analyzed by HPLC over a gradient of 5% B to 95% B over 15 min at a flow rate of 1 mL/min through a Viva C18 column. Peptide elution was monitored by absorbance measurements at 210 nm. After correcting for drift in the baseline, the area of the H3 elution peak was determined in MATLAB using the trapezoid method. Cell Culture and Microinjection. NIH/3T3 cells were maintained in DMEM with 10% fetal bovine serum, 100 μg/mL penicillin, and 100 μg/mL streptomycin. The day prior to microinjection, 25,000 cells were plated into each well of a twowell slide. 40 μL polyplex solutions were made as described and mixed with 10 μL of a 21 mg/mL solution of 70 kDa dextran conjugated to Texas Red in H2O. The polyplex−dye solutions were subsequently centrifuged at 5000g for 5 min to remove any aggregates that might clog the injection needle. Polyplex− dye solutions were backloaded into Femtotips II with Femtoloaders, and microinjections were performed with a semiautomatic microinjection setup, using a Femtojet and Injectman NI 2 (Eppendorf). An injection pressure of 120 hPa was used for a duration of 0.3 s, and a compensation pressure of 30 hPa was maintained on the needle to ensure that media would not diffuse into the injection solution. Cell injection was 1043

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mixtures were agitated for 1 h at 4 °C, after which the beads from each sample were removed, washed, and added to 30 μL of PBS containing 0.1% sodium dodecyl sulfate (SDS), 3 μL of Laemmli buffer, and 3 μL of β-mercaptoethanol. Laemmli− bead solutions were boiled for 5 min, the beads were removed, and the bead-free solutions were analyzed via SDS− polyacrylamide gel electrophoresis (SDS−PAGE) on a 12% acrylamide gel followed by transfer onto polyvinylidene fluoride (PVDF) membranes. Western blotting was performed according to standard protocols, with blocking solutions of 5% BSA in Tris buffered saline containing Tween-20 (TBST). The anti-HBO1 rabbit polyclonal antibody and a donkey antirabbit polyclonal antibody conjugated to HRP were used to probe the membranes. The HRP was activated with the Amersham ECL Plus Western Blotting Detection Kit (GE Healthcare) and imaged on a Typhoon 9400 Variable Mode Imager (GE Healthcare). Statistical Analysis. A Student’s t test was utilized to determine significant differences in sample populations.

observed on a Nikon Eclipse TE2000-U microscope (Tokyo, Japan) under ambient conditions. Approximately 100 cells were injected within 15 min in order to minimize cell exposure to atmospheric conditions. For time-lapse imaging analyses, the microinjected cells were immediately placed on a climate-controlled stage (37 °C and 5% CO2). Image acquisition was performed with a Zeiss Axiovert 2000 microscope (Thornwood, NY), with images obtained every 5 min over a 6 h period starting 30 min after the injections were made. For transfection analyses, the microinjected cells were transferred back into the incubator and analyzed at later times via fluorescence microscopy on a Leica DMI 6000B inverted fluorescence microscope (Wetzlar, Germany). Delay Time Analyses and Unpackaging Model. The GFP intensities of individual cells were measured as a function of time (Figure 1a) and the integrated cell intensities calculated by quantitative image analysis with ImageJ (NIH, Bethesda, MD) (Figure 1b). Because it is well-known that the volume of a microinjection varies widely from cell to cell,39 and therefore, that the number of microinjected plasmids will vary from injection to injection, integrated fluorescence measurements were normalized from 0 to 1 using the equation normalized GFP intensity =

I(t ) − Imin Imax − Imin

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RESULTS H3-Based Peptides Form Compact, Nanoscale Polyplexes with pDNA. To ensure that the H3-based peptides could efficiently complex DNA, and to characterize the structures of the resulting polyplexes, we self-assembled polyplexes from GFP-encoding pDNA and the H3 tail peptides, and characterized the assembled H3−pDNA polyplexes by gel electrophoresis and DLS (Figure 2). Gel electrophoresis assays indicated that, at N:P ratios of 1 or less, the pDNA interacted with the H3 peptides but was not fully complexed, as evidenced by the presence of migrating DNA bands of reduced mobility (Figure 2a). As the N:P ratio increased above 1, the DNA bands were retained in the well and lost intensity, indicating that the pDNA was entirely complexed and tightly compacted by the H3 peptides. The fluorescence of the pDNA in the wells decreased as the N:P ratio increased up until N:P = 5, at which point the well fluorescence was constant. DLS analyses were consistent with the gel electrophoresis assays (Figure 2b). As the N:P ratio increased, the hydrodynamic diameters of the polyplexes initially decreased, demonstrating the increased complexation state at N:P = 2.5 as compared with N:P = 1.25. H3−pDNA Polyplexes Exclude Serum Nucleases at N:P Ratios Greater than 1.25. Because tightly packaged polyplexes are known to exclude both nucleases and the transcriptional machinery from the packaged pDNA, we next examined the accessibility of the pDNA to proteins via nuclease degradation assays (Figure 2c). As seen in the first lane, the pDNA was present as a mixture of the supercoiled and open circular isoforms. When free pDNA was incubated with DNase I, the pDNA was degraded, as seen through the elimination of the bands in lane 2. In contrast, when H3−pDNA polyplexes were incubated with DNase I, the pDNA was recovered, as seen in lanes 3−6. To ensure that the H3 did not disassociate nonspecifically from the pDNA at physiologically relevant ionic strength, the same experiment was repeated at physiological ionic strength of 150 mM. As seen in lanes 9−12, the pDNA in the polyplexes was protected from degradation, demonstrated by the retention of fluorescence after DNase treatment. Furthermore, given that multiprotein transcriptional complexes are significantly larger than serum nucleases, these data suggested that the transcriptional machinery would be unable to access the pDNA in polyplexes formulated at N:P ratios greater than 1.25.

(1)

where I(t) is the GFP intensity at time t, and Imin and Imax are the minimum and maximum GFP intensity values, respectively. A subset of the normalized data is depicted in Figure 1c. Delay time calculations on the normalized intensity data were performed through a least-squares regression on the linear region of these data. The linear region was chosen by differentiation of the intensity function with respect to time, to determine when the normalized intensity was approximately constant (Figure S1b in the Supporting Information). The xintercept was determined from the linear regression. The unpackaging model for the DNA polyplexes was implemented using MATLAB version R2008b (Mathworks, Natick, MA). Specifically, ODE45 was used to numerically solve equations, as these were non-stiff equations, and lsqcurvefit was used to determine the rate constants in the model. MATLAB code can be found in the Supporting Information. A sensitivity analysis was separately performed for each sample as previously described,40 by varying the initial concentration of pDNA or polyplex and examining the relative changes in the modeled rate parameter. The initial concentration of pDNA or polyplex was varied up to 10%, and samples were rejected if the sensitivity of the rate constant was greater than 10−4. HBO1 Pull-Down Assay. H3−biotin−pDNA and biotinylated-PEI−pDNA polyplexes were made as previously described. 43.8 μL of 20 mM HEPES and 1.5 μL of a 100 mM phenylmethylsulfonyl fluoride (PMSF) solution in ethanol were sequentially added to 100 μL of each polyplex solution. The resulting solutions were placed on ice for 10 min, and 6.2 μL of HBO1-enhanced mouse cell lysate was added to each sample. Samples were then gently mixed and agitated for 4 h at 4 °C. M-280 streptavidin-coated Dynabeads were used for pulldown of the biotinylated polyplexes, according to the manufacturer’s protocols. For each sample, 25 μL of Dynabeads was placed in 175 μL of PBS, and subsequently, the beads were washed and added to the polyplex/cell lysate solution. These 1044

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Figure 2. Physical characterization of the H3−pDNA polyplexes. (a) A representative agarose gel shows the level of pDNA complexation at varying N:P ratios via ethidium bromide staining. Lane 1 is the gWIZ plasmid alone, and the remaining lanes are the indicated polycations complexed with pDNA at the indicated N:P ratios. (b) The average hydrodynamic diameters of H3K4Me3−pDNA (solid) and H3K4− pDNA (open) polyplexes as determined by dynamic light scattering. The data represent the mean ± standard error (n = 3). (c) A representative gel demonstrating the nuclease stability of the pDNA within various H3−pDNA polyplexes at ionic strengths of 20 mM and 150 mM as determined by serum incubation and agarose gel electrophoresis. Lanes 1 and 7 are pDNA incubated without serum, lanes 2 and 8 are pDNA incubated with serum, lanes 3 and 9 are H3K4−pDNA polyplexes at an N:P of 1.25, lanes 4 and 10 are H3K4−pDNA polyplexes at an N:P of 2.5, lanes 5 and 11 are H3K4− pDNA polyplexes at an N:P of 5, and lanes 6 and 12 are H3K4− pDNA polyplexes at an N:P of 10.

Figure 3. Analysis of H3−pDNA polyplex composition. (a) The quantity of H3 peptide in the polyplex (black) or free in solution (white) in polyplex solutions containing 2 μg of DNA. The amount of peptide was quantified by integration of the HPLC absorbance spectra. (b) The number of peptides per plasmid for H3K4Me3 (black) and H3K4 (white) polyplexes. The values shown represent the mean ± standard error (n = 3); * indicates a statistically significant difference between the indicated values (p < 0.05).

amount of H3 peptide found in solution, whereas at N:P > 2.5, the fraction of free peptide in solution increased substantially with increasing N:P ratio. A significantly larger number of peptides were found in the polyplexes at N:P = 5 as compared with at N:P = 2.5 (Figure 3b). Lastly, consistent with gel electrophoresis studies, there was no difference between the abilities of the H3K4Me3 and H3K4 peptides to bind to pDNA, as approximately the same number of each peptide was associated with the pDNA at a given N:P ratio. H3−pDNA Polyplexes Transfect Cells at Significantly Higher Levels than PEI-Based Polyplexes. To explore the effects of both the polyplex packaging state and the active trimethylation motif on the gene transfer activity, we microinjected free pDNA or the polyplexes directly into the nuclei of NIH/3T3 cells and evaluated the efficiencies with which the injected solutions transfected cells via fluorescence microscopy (Figure 4). When free pDNA was microinjected, approximately 90 ± 2% of the cells expressed GFP. In contrast, when PEI−pDNA polyplexes were microinjected, significantly fewer cells expressed GFP. Transfection was maximal at an N:P ratio of 10, with 49 ± 4% of the cells expressing GFP. PEI− pDNA polyplexes at an N:P ratio of 2.5 were too large to be microinjected without clogging the needle, and thus were not tested.

Trimethylated and Non-Trimethylated H3 Peptides Associate Similarly with pDNA. To further elucidate the complexation state of the polyplexes at the various N:P ratios, the composition of the H3−pDNA polyplexes was determined by HPLC analysis. Polyplexes were formed, and subsequently, any uncomplexed H3 peptides were separated from the polyplexes by centrifugal filtration and quantified by HPLC analysis (Figure 3). A calibration curve was created with samples that were not complexed by pDNA but otherwise were treated identically. The number of moles of H3 within the polyplexes was determined based on the difference in the areas of absorbance of the H3 samples without pDNA and the H3 samples that were complexed with pDNA. As seen in Figure 3a, the number of moles of H3 peptide within the polyplexes increased as a function of increasing N:P ratio up to a maximum of ∼2.2 nmol of peptide per 2 μg of pDNA. Furthermore, at an N:P ratio of 2.5, there was a negligible 1045

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Figure 4. Transfection efficiency post-nuclear microinjection. The mean transfection efficiency for H3K4Me3−pDNA polyplexes (filled), H3K4−pDNA polyplexes (open), PEI−pDNA polyplexes (thatched), and free pDNA (diagonal), 5 h postinjection. The triangle indicates that the PEI−pDNA polyplexes were examined at an N:P ratio of 6. * indicates a statistically significant difference (p < 0.05). Error bars represent the standard error of the measurements.

In contrast to the PEI−pDNA polyplexes, the H3-based polyplexes were efficient transfection agents. The H3K4Me3− pDNA polyplexes transfected 86 ± 4% of the cells at an N:P ratio of 2.5. As the N:P ratio increased, the percentage of cells that were successfully transfected with these polyplexes decreased, presumably due to the increased extent of complexation at higher N:P ratios (Figure 3). Additionally, it appeared that the trimethylation on the H3 peptide enhanced the overall activity of the polyplexes at low N:P ratios, as the percentage of successfully transfected cells was significantly higher (P < 0.05) for the H3K4Me3−pDNA polyplexes in comparison with the H3K4−pDNA polyplexes at an N:P ratio of 2.5. H3−pDNA polyplexes were not analyzed at N:P ratios of 1.25 or below because they clogged the needle due to their large size. The H3K4Me3 Motif Facilitates Rapid Gene Expression within the Nuclei of Cells. To further analyze the effects of the trimethylation state of the peptide, we next asked whether the H3K4Me3 motif could initiate polyplex unpackaging and the recruitment of the transcriptional machinery, and thereby accelerate the rate of gene expression in microinjected cells. We thus evaluated the kinetics of GFP expression in microinjected cells via time-lapse fluorescence microscopy. To facilitate the quantification of expression rates based on data from these experiments, we developed a simple, noncompartmentalized cellular model based on theory of the dynamic behavior of linear first-order systems.41 Because the presence of the trimethylated H3 peptides was expected to accelerate the rate of onset of GFP expression, we calculated and quantitatively compared the delay times until GFP protein production was observable as the x-intercepts from the linear regimes in GFP expression for each pDNA formulation (Figure S1 in the Supporting Information and Figure 5a). As the rates for transcription and translation should be similar from cell to cell, any variations in delay time should be due to differences in the nuclear activity of the transfection agent. As seen in Figure 5b, GFP was first observable approximately 104 ± 6 min after microinjection with uncomplexed pDNA. GFP took approximately 50% longer to become observable

Figure 5. Delay time analysis. (a) An idealized representation of GFP production based on the systems approach to determine the delay time, in which the GFP content in the cell is depicted as a function of time on the graph. The linear regime of the graph (overlaid line) was used to determine the onset of observable protein production. The difference between the x-intercept of the linear regime (dash−dot line) and the time at which the injection occurred (dashed line) was used as the delay time until observable protein production. (b) The delay time until observable protein production for H3K4Me3−pDNA polyplexes (filled), H3K4−pDNA polyplexes (open), PEI−pDNA polyplexes (thatched), and free pDNA (diagonal), where the error bars represent the standard error in the measurements. The triangle indicates that PEI was run at an N:P ratio of 6. * indicates a statistically significant difference (p < 0.05).

when PEI−pDNA polyplexes were microinjected, suggesting that the complexation of pDNA by cationic macromolecules such as PEI indeed inhibited transcriptional activity via steric hindrance. Similar effects were observed when the cells were microinjected with the H3K4−pDNA polyplexes at a variety of N:P ratios. In contrast, at N:P ratios of 2.5 and 10, the microinjected H3K4Me3−pDNA polyplexes exhibited significantly shorter delay times that were comparable to the delay times observed with uncomplexed pDNA. The different behaviors of the H3K4Me3−pDNA polyplexes and the H3K4−pDNA polyplexes at these N:P ratios suggested an active role for trimethylation in promoting gene expression. To determine whether the observed differences in delay times were correlated with differences in the rates of polyplex unpackaging and transcriptional activation, we developed a mass action kinetic model of DNA unpackaging and the subsequent downstream processing steps leading to gene expression based upon previously developed nonviral gene delivery models.42−44 Each processing step was modeled as first-order, and unpackaging and transcriptional activation were 1046

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Table 1. kunpack [min−1]a for H3−pDNA Polyplexes N:P ratio

2.5

5

10

H3K4Me3−pDNA H3K4−pDNA PEI−pDNA

>1b 0.020 ± 0.002

0.032 ± 0.004 0.052 ± 0.009 0.022 ± 0.001c

>1b 0.050 ± 0.008 0.08 ± 0.02

a kunpack values represent the predicted values ± 95% confidence intervals. b>1 indicates that the value was highly sensitive to initial guess. cPerformed at an N:P ratio of 6.

bind to HAT protein complexes, pull-down assays were performed with pDNA polyplexes containing biotinylated peptides and HBO1-enhanced cell lysates. After validating that biotinylation did not interfere with complexation for either the H3- or PEI-based polyplexes (Figure S3 in the Supporting Information), the pDNA polyplexes were incubated with the cell lysates and the resulting interactions between the polyplexes and HBO1 were analyzed via Western blotting (Figure 7). Both the trimethylated and the non-trimethylated

Figure 6. pDNA polyplex reaction network. The modeled reaction pathway for pDNA polyplex unpackaging, pDNA transcription, and mRNA translation. The underlined rate constants were determined through model fits implemented in MATLAB; all other rate constants were found in the literature.

lumped into a single kinetic parameter (Figure 6), resulting in expressions describing the concentration profiles for pDNA and other species: d[complex] = −k unpack[complex] dt

(2)

d[DNA] = k unpack[complex] dt

(3)

d[mRNA] = k mRNA[DNA] − k m,deg[mRNA] dt

(4)

d[protein] = k prot[mRNA] − k p,deg[protein] dt

(5)

where “complex” represents microinjected pDNA polyplexes, “DNA” represents unpackaged pDNA, “mRNA” and “protein” are GFP-encoding mRNA and the GFP protein, respectively, kunpack is the pDNA unpackaging rate constant, kmRNA and kprot are the rate constants at which mRNA and protein are being produced, respectively, and km,deg and kp,deg are the rate constants at which mRNA and protein are being degraded, respectively. The rate constants for mRNA and protein degradation were found in the literature.45 It was assumed that there was no pDNA degradation in the nucleus, consistent with other models found in the literature43,46 and with the absence of known nuclear DNases. kmRNA and kprot were determined to be 0.04 and 0.3 min−1, respectively, by using data obtained from samples in which free (uncomplexed) pDNA was microinjected, as the unpackaging rate constants could be eliminated for these samples. The values for kunpack were then fit by least-squares estimates for each of the various polyplexes (Figure S2 in the Supporting Information), which demonstrated that, as expected, unpackaging and transcriptional activation of the H3K4Me3−pDNA polyplexes were generally extremely rapid in comparison with the same activities for either the H3K4−pDNA polyplexes or the PEI−pDNA polyplexes (Table 1). The value of kunpack for the H3K4Me3− pDNA polyplexes at an N:P ratio of 5 was significantly lower than the values of kunpack for the H3K4Me3−pDNA polyplexes at N:P ratios of 2.5 or 10, which was reflective of the observed differences in delay times between the various polyplexes. H3−pDNA Polyplexes Associates with HAT Protein Complexes. To determine whether the increased transfection efficiencies and protein expression rates caused by the H3containing polyplexes were caused by their enhanced abilities to

Figure 7. HBO1 pull-down assay. Western blot against HBO1 following pull-down of H3−biotin−pDNA polyplexes, biotinylatedPEI−pDNA polyplexes or free H3−biotin peptides after incubation with HBO1-enhanced cell lysates. Lane 1 is a sample of lysate proteins that were not incubated with the Dynabeads. Lanes 2−5 contain samples of lysate proteins that associated with the indicated polyplexes or peptides during pull-down with the Dynabeads. Lane 6 contains a sample of lysate proteins that associated nonspecifically with polyplexfree and peptide-free Dynabeads. Lanes 7 and 8 contain samples of lysate proteins that associated with the indicated polyplexes during pull-down with the Dynabeads.

polyplexes pulled down HBO1, whereas polyplex-free beads did not, suggesting that H3 peptide-specific interactions with HBO1 were occurring, and potentially capable of enhancing the gene transfer activities of the polyplexes. HBO1 pull-down was not significantly greater for the H3K4Me3−pDNA polyplexes as compared with the H3K4−pDNA polyplexes. We presumed that any differences in HBO1 interaction between the two H3 motifs were not detectable by the pulldown assay. To confirm that the interaction between the H3− pDNA polyplexes and HBO1 was not caused by nonspecific electrostatic interactions, the capability of biotinylated-PEI− pDNA polyplexes to pull down HBO1 was tested. As seen in lanes 7 and 8 of the Western blot shown in Figure 7, there was no significant interaction between the biotinylated-PEI−pDNA polyplexes and HBO1.

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DISCUSSION Significant evidence suggests that the tight, electrostatic condensation of chromosomal DNA or pDNA reduces DNA 1047

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packaged prior to delivery, to the extent that they could prevent DNA degradation by DNases, both the trimethylated and nontrimethylated H3 polyplexes were better able to transfect cells than the PEI−pDNA polyplexes. These higher transfection efficiencies might be partly caused by the slight differences in the electrostatic interactions between the cationic complexation agents and the pDNA, as heparin displacement assays indicated that less heparin was required to unpackage the H3−pDNA polyplexes in comparison to the PEI−pDNA polyplexes (Figure S4 in the Supporting Information). Gel electrophoresis experiments also indicated minor differences in the degree of polyplex packaging between the H3-based polyplexes and the PEI−pDNA polyplexes; the ethidium bromide fluorescence was entirely absent when PEI was used to complex the pDNA due to ethidium bromide exclusion,36 whereas there was weak fluorescence in the lanes containing the H3-based polyplexes, even at an N:P ratio of 10 (Figure 2a). These minimal differences in the extent of packaging are consistent with other reports in the literature, which demonstrate that shorter polycations package pDNA less efficiently, and bear a stronger thermodynamic tendency to dissociate from it.6 However, the H3 peptide was still associated with pDNA at high ionic strengths, as indicated by the recovery of pDNA after nuclease digestion (Figure 2c), indicating that the pDNA should still be tightly packaged when introduced into the nucleus of a cell. The transfection experiments also provided evidence for an active role for the H3 peptides and the trimethylation motif in promoting unpackaging and transcriptional activation, as differences were observed in the transfection efficiencies of the trimethylated and non-trimethylated polyplexes. At low N:P ratios, the H3K4Me3−pDNA polyplexes transfected significantly more cells than the H3K4−pDNA polyplexes, most likely due to the ability of the H3K4Me3−pDNA polyplexes to promote the recruitment of nuclear effector complexes such as HBO1 HAT and NURF. The recruited HAT complexes would be expected to hyperacetylate the H3 peptide, as all nonmodified lysines on this peptide can be acetylated.53 Acetylation would screen the electrostatic interactions between the H3 peptide and the pDNA, which in turn would allow the transcriptional machinery to bind directly to the pDNA. We would expect to see this effect most prominently at the lower N:P ratios, when screening one residue would have the greatest impact on the overall N:P ratio. In addition to impacts on the overall transfection efficiency, we expected that trimethylation would enhance the rate of transcription, with observable effects on the onset and initial rate of gene expression. Thus, to determine whether trimethylation induced immediate enhancements in transcriptional activation, we sought to directly measure the rates of gene expression following microinjection with the various polyplexes. Our data suggested that the H3K4Me3 peptide was capable of accelerating the onset of gene expression by these cells, as, in general, the delay times until expressed protein was detectable were significantly longer when polyplexes lacking the H3K4Me3 motif were used. Indeed, the delay times associated with the H3K4Me3−pDNA polyplexes at N:P ratios of 2.5 and 10 were comparable to those of naked pDNA, suggesting that the H3K4Me3 motif promoted the essentially immediate access of the pDNA to the transcriptional machinery. We next confirmed that these differences in delay time corresponded to increases in the rates at which the pDNA was unpackaged/activated (Figure 5b) by modeling unpackaging and downstream processing with first-order mass action

transcription by sterically restricting DNA accessibility, yet in the case of lipoplexes and polyplexes, tight packaging is necessary during the extranuclear steps in the gene delivery process. We have developed gene delivery materials containing post-translationally modified histone H3 tail peptides that have been linked to chromosomal DNA activation, with the goal of recapitulating the natural unpackaging and transcriptional activities of these peptides on synthetic pDNA. In this work, we demonstrate that the H3 tail peptides tightly complexed pDNA through electrostatic interactions, yet initiated efficient transcription within the nucleus. H3-based polyplexes interacted with nuclear effector proteins associated with chromatin activation, suggesting that they play an active role in stimulating transcription. We thereby provide new evidence supporting polyplex unpackaging/activation as a gene delivery barrier, and the first example of a role for epigenetic modifications in promoting nonviral gene delivery. We demonstrated that the H3 peptides were capable of complexing pDNA, and that the methylation state of the peptide did not affect complexation (Figure 2 and 3). This was anticipated because methylation does not alter the charge state of lysine, and it did not significantly alter the number of peptides per plasmid (Figure 3b). Complexation substantially protected the pDNA from nuclease digestion, as the band intensities of recovered pDNA were similar to the original pDNA band intensities (Figure 2c, lane 7 vs lanes 9−12). However, the bottom pDNA band in the polyplex digests was retarded in comparison to the bottom band for nondigested pDNA. This may potentially be due to partial plasmid linearization, indicating slight access of the pDNA while in complex with the H3 peptide. The appearance and positioning of these bands were similar to multiple examples in the literature, and the presence of both bands is generally accepted to show that the pDNA is intact and primarily inaccessible to nucleases.36,47−49 To confirm that tightly packaged polyplexes inhibited pDNA transcription, and to ask whether the methylation state of the H3 peptide would alter its nuclear activity and promote pDNA unpackaging and activation, we performed nuclear microinjection experiments with our polyplexes in NIH/3T3 cells. As expected, we observed that PEI complexation hindered the transcription of the pDNA, as the transfection efficiencies were at least 40% lower when cells were microinjected with PEI− pDNA polyplexes as compared with naked pDNA. These differences were similar over a range of N:P ratios, as PEI− pDNA polyplexes formulated at an N:P ratio of 10 exhibited similar transfection activities as those formulated at N:P = 6. The effects of N:P ratio on PEI-mediated transfection were consistent with the literature, which demonstrates that an N:P ratio of 6 achieved nearly optimal transfection efficiency while minimizing cell death.50,51 Furthermore, these activity differences between packaged DNA and naked pDNA are generally consistent with differences observed previously in the literature. For example, Pollard et al. have shown that 40% of COS-7 cells were successfully transfected 4 h postnuclear injection with PEI−pDNA polyplexes, whereas 60% of the cells injected with naked pDNA were successfully transfected.52 When we compared the transfection efficiencies achieved by the nuclearly injected PEI−pDNA polyplexes with those of the H3K4Me3−pDNA and the H3K4−pDNA polyplexes, it was clear that the extent to which the polyplexes were transcribed depended strongly on the delivery vehicle. While both PEI− pDNA polyplexes and the H3-based polyplexes were tightly 1048

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promote more substantial and rapid activation.25,26,54 That these early effects did not translate into sustained differences in transfection is reasonable given the observed interactions between non-trimethylated polyplexes and nuclear effectors. These interactions would cause a slower activation and expression process, consistent with the elevated levels of transfection for both types of H3-based polyplexes in comparison with PEI polyplexes. This work is the first to combine nuclear microinjection techniques with model-based quantification of gene expression kinetics and, as such, newly enables quantification of both the delay times and rates of gene expression by pDNA and polyplexes. These direct measurements of gene expression provide key evidence supporting unpackaging as a gene delivery barrier and, furthermore, provide the first example in which epigenetic sequences have been successfully utilized to stimulate synthetic gene delivery. Other work in our laboratory has shown that these enhancements in gene expression levels translate into enhancements in cellular transfection efficiencies.55 Thus, collectively, our studies indicate an important role for histone-based peptides in nonviral gene delivery, with particular relevance to therapeutic scenarios in which the rapid onset of gene expression is critical. Through the incorporation of the H3K4Me3 motif in nonviral gene delivery vehicles, transcriptional activation and, ultimately, overall efficacy may be greatly improved.

kinetics (Figure 6). The high values of kunpack for the H3K4Me3 complexes at N:P ratios of 2.5 and 10 were attributed to changes in polyplex complexation state and peptide accessibility. As demonstrated through the determination of polyplex composition, the H3-based polyplexes became more tightly complexed as the N:P ratio increased up to N:P = 5 (Figure 3b). These changes in complexation are consistent with the increase in delay time from N:P = 2.5 to N:P = 5. At N:P = 5, the polyplexes had reached their maximal compaction state. Thus, at N:P > 5, additional peptides would be surfaceassociated and able to recruit HBO1 and other nuclear effectors, consistent with the observed reduction in delay time and increase in kunpack. Because we expected that the enhanced nuclear activities of the H3-based polyplexes, and in particular those of the H3K4Me3−pDNA polyplexes, were conferred by their capacity to interact with various nuclear protein complexes such as the HBO1 HAT, we asked directly whether the polyplexes interacted with HBO1 via pull-down assays with HBO1enhanced cellular extracts. As expected, these assays indicated that the H3K4Me3−pDNA polyplexes interacted with HBO1 at levels sufficient for pull-down. Surprisingly, our assays indicated that the levels of HBO1 pull-down were similar for both the trimethylated and the non-trimethylated polyplexes. We attribute the similar HBO1 pull-down behavior of the trimethylated and non-trimethylated polyplexes to difficulties in sensitive quantification by immunoprecipitation and Western blotting. Specifically, although there are differences in affinity, both the trimethylated and non-trimethylated peptides interact with the PHD binding region in HBO1.54 In the presence of a cell lysate solution that was engineered to contain high levels of HBO1, it is reasonable to expect that both peptide variants would become saturated, and would thus exhibit similar pulldown behavior. It is unlikely that the HBO1−H3K4 binding event was purely nonspecific (i.e., electrostatic), as we did not observe any interaction of the HBO1 complex with biotinylated-PEI/pDNA polyplexes (Figure 7).

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ASSOCIATED CONTENT

S Supporting Information *

Information that further explains how the analysis of the microinjection data was performed and the raw and fitted data of the increase of GFP intensity over time. Further analysis to confirm that biotinylation did not significantly alter complexation and a brief investigation of nonspecific unpackaging effects. All MATLAB codes. This material is available free of charge via the Internet at http://pubs.acs.org.

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CONCLUSION We have provided the first evidence that the epigenetic code can be utilized to promote enhancements in both the rate and efficiency of transfection by exogenous pDNA in a nonviral gene delivery vehicle. Histone H3 peptides formed pDNA polyplexes that were tightly packaged and avoided nuclease degradation in physiological saline prior to nuclear entry. Within the nucleus, trimethylated H3-based polyplexes stimulated rapid transcription, and H3 polyplexes both with and without trimethylation transcribed substantially more cells than standard PEI-based polyplexes. These enhancements in the rate and level of transcription by the H3 polyplexes are likely a combination of passive and active effects. The H3 peptides were more easily displaced from pDNA by counterions, as compared with PEI. In addition, H3-based polyplexes interacted with nuclear effectors associated with transcriptional activation, whereas PEI-based polyplexes did not, indicating that active recruitment likely contributed to the differences in activity. Although minimal differences were observed between the H3K4 and H3K4Me3 polyplexes in transfection efficiency, clear and significant differences were found in the onset and rate of gene expression. These early effects are consistent with the stronger affinity of the trimethylated polyplexes for nuclear effectors containing key PHD recognition motifs, which would

AUTHOR INFORMATION

Corresponding Author

*University of Delaware, Department of Chemical Engineering, Colburn Laboratory, 150 Academy St., Newark, DE 19716. Phone: (302) 831-8072. Fax: (302) 831-1048. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by a grant from the National Science Foundation (DMR-0746458) to M.O.S. 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 thank Ulhas Naik for the generous use of microinjection equipment. Furthermore, we thank Kristi Kiick for useful discussions, and Michael Salciccioli for help with the modeling of polyplex unpackaging.

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