Express in Vitro Plasmid Transfection Achieved with 16+ Asymmetric

Feb 18, 2016 - Asymmetric cationic amino acid-based dendrimers are highly branched chemically derived gene vectors developed to transport cargo such a...
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Article pubs.acs.org/journal/abseba

Express in Vitro Plasmid Transfection Achieved with 16+ Asymmetric Peptide Dendrimers Prarthana V. Rewatkar,† David P. Sester,‡ Harendra S. Parekh,*,† and Marie-Odile Parat*,† †

School of Pharmacy, The University of Queensland, 20 Cornwall Street, Woolloongabba, Queensland 4102, Australia School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Queensland 4072, Australia

‡

S Supporting Information *

ABSTRACT: Asymmetric cationic amino acid-based dendrimers are highly branched chemically derived gene vectors developed to transport cargo such as plasmid DNA across the plasma membrane. We have previously demonstrated their propensity to enter cells that form caveolae, driven by positive charge density and promoted by arginine head groups. Caveolae are plasma membrane subdomains serving a number of cellular functions including endocytosis. Their formation requires membrane proteins (caveolins) and cytoplasmic proteins (cavins), so that gene disruption of either caveolin-1 or cavin-1 (also known as PTRF, i.e., polymerase I and transcript release factor) results in caveola deficiency. Here we evaluated the ability of a 16+ charged asymmetric arginine dendrimer to transfect plasmid DNA into cultured cells. We unveiled efficient transfection efficiencies (≥30%) 24−48 h after exposing the cells to dendrimer/pDNA complexes for only 5 min. Using wild type (WT) and caveolin-1 or PTRF gene-disrupted, i.e., caveola-deficient mouse embryo fibroblasts, we further show that caveolae promote pDNA transfection by 16+ charged asymmetric arginine dendrimers. KEYWORDS: Caveolae, peptide dendrimer, pEGFP-N1, transfection, Caveolin-1, PTRF/Cavin-1, arginine

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mouse embryo fibroblasts (iMEFs) harboring or devoid of caveolae. The dendrimer was complexed with plasmid DNA encoding the expression of green fluorescent protein (pEGFP) to transfect iMEF wild type, iMEF cav-1(−/−) and iMEF PTRF(−/−) cells. A nonviral, cationic lipid-based reagent widely used to transfect cells with plasmid DNA, namely Lipofectamine 3000, was used as a reference. Green fluorescence was employed as the readout for transfection efficiency, visualized using fluorescence microscopy and quantified via flow cytometry analysis.

INTRODUCTION Gene therapy is a strategy of increasing significance for the treatment of various disorders. Successful gene delivery, however, depends on the development of delivery vectors devoid of toxicity and able of high rates of transfection. To date, a large body of research has focused on using viral vectors such as adenoviruses and retroviruses to deliver genes because of their high transfection efficiency. However, a fundamental complication associated with their use has included the potential for immunogenicity and anaphylaxis.1,2 In contrast, nonviral vectors such as low generation asymmetric cationic dendrimers do not elicit unwanted immune responses, while readily forming complexes with genetic material and retaining their ability to cross various cellular barriers.3 Multiple studies have tested, using pharmacological inhibitors, whether dendrimers alone or complexed with DNA use caveolae as an endocytosis route.4−7 We have previously demonstrated using a panel of biotinylated cationic asymmetric peptide dendrimers, that the 16+ arginine dendrimer was internalized promptly, efficiently, and preferentially by cells forming caveolae.8 In the current study, we optimized and evaluated the use of this 16+ charged asymmetric arginine dendrimer for express plasmid DNA (pDNA) transfection. To evaluate the role of caveolae, we used immortalized © 2016 American Chemical Society

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MATERIALS AND METHODS

Reagents. Peptide grade N,N-dimethylformamide (DMF) was purchased from Merck (NSW, Australia). All fluorenylmethyloxycarbonyl (Fmoc) amino acids and Rink amide resin (200−400 mesh) were obtained from NovaBiochem (NSW, Australia). N,N-Diisopropylethylamine (DIPEA), trifluoroacetic acid (TFA), triisopropylsilane (TIPS), O-Benzotriazole-N,N,N′,N′-tetramethyl-uronium-hexafluoro-phosphate (HBTU), dichloromethane (DCM), piperidine, and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (Castle Hill, Received: January 20, 2016 Accepted: February 18, 2016 Published: February 18, 2016 438

DOI: 10.1021/acsbiomaterials.6b00033 ACS Biomater. Sci. Eng. 2016, 2, 438−445

Article

ACS Biomaterials Science & Engineering

Figure 1. A 16+ asymmetric arginine dendrimer efficiently transfects pDNA into cells following an exposure time of 5 min. WT MEFs were seeded at 10 000 cells per well in 6-well plates and incubated in cell culture medium with 5% serum for 24 h. Cells were exposed to pDNA alone (2 μg/mL), to Lipofectamine 3000/pDNA (2 μg/mL) complex, or to 16+ asymmetric arginine dendrimer (50 μg/mL)/pDNA (2 μg/mL) complex in serum- and antibiotic-free medium for 5 min. The transfection mixture was removed and cells were incubated in 2.5% (v/v) serum medium for 4−48 h and analyzed using flow cytometry. A) Results are reported as % transfection efficiency and shown as mean ± SEM (n = 3 separate experiments); ns, not significant, ****p < 0.0001. (B) Results are reported as median fluorescence intensity per cell as quantified using FlowJo, and expressed as arbitrary units (AU). Shown are mean ± SEM (n = 3 separate experiments); ns, not significant. (C) Representative flow-cytometric data of EGFP expression at 48 h post transfection for untreated, pDNA Alone, Lipofectamine 3000/pDNA and dendrimer/pDNA samples. (D) Representative bright-field and fluorescence microscopy images of cells 48 h post transfection exposed for 5 min to pDNA alone, Lipofectamine 3000/pDNA or dendrimer/DNA as indicated. Bar represents 50 μm. 100 IU/mL penicillin, and 100 μg/mL streptomycin at 37 °C in a humidified atmosphere with 5% CO2. Synthesis of Asymmetric Arginine-Terminated Peptide Dendrimer. An asymmetric 16+ arginine dendrimer was synthesized as previously described8 using Fmoc solid-phase peptide synthesis. Briefly, the dendrimer was constructed on an insoluble solid support (Rink amide resin) followed by sequential coupling of Nα-Fmoc protected amino acids, whereby glycine was employed as an initial spacer residue, followed by sequential Lys residues constituting the dendrimer core, and terminating with Arg residues at the periphery. To

NSW, Australia). D-Biotin was acquired from Shen Zhen Inno Syn Biotech Co., Ltd. (Shenzhen, China). Dulbecco’s modified Eagle’s medium (DMEM), penicillin/streptomycin solution, trypsin, fetal bovine serum (FBS), and Lipofectamine 3000 were obtained from Invitrogen (Life Technologies, Mulgrave, VIC, Australia). Cell Culture. Immortalized Mouse Embryo Fibroblasts (iMEFs) isolated from wild type (WT), caveolin-1 gene-disrupted (Cav-1 (−/−)), and PTRF gene-disrupted (PTRF (−/−)) mice were maintained in DMEM medium supplemented with 5% (v/v) FBS, 439

DOI: 10.1021/acsbiomaterials.6b00033 ACS Biomater. Sci. Eng. 2016, 2, 438−445

Article

ACS Biomaterials Science & Engineering

Figure 2. Transfection efficiency of 16+ asymmetric arginine dendrimer/pDNA complexes applied for 5 min is equivalent to that of Lipofectamine 3000/pDNA complexes applied for 2.5 h. WT MEFs were seeded at 10 000 cells per well in 6-well plates and incubated in cell culture medium with 5% serum for 24 h. Cells were exposed for 2.5 h to serum- and antibiotic-free medium (control), to pDNA alone (2 μg/mL), to Lipofectamine 3000, or to Lipofectamine 3000/pDNA (2 μg/mL) complex. Alternatively, cells were exposed for 5 min to 16+ asymmetric arginine dendrimer (50 μg/mL), or to 16+ asymmetric arginine dendrimer (50 μg/mL)/pDNA (1 or 2 μg/mL) complex in serum- and antibiotic-free medium as indicated. The transfection mixture was removed, cells were incubated in 2.5% serum (v/v) medium for 48 h and analyzed using flow cytometry. A) Results are reported as % transfection efficiency and shown as mean ± SEM (n = 3 separate experiments). (B) Results are reported as median fluorescence intensity per cell as quantified using FlowJo, and expressed as arbitrary units (AU). Shown are mean ± SEM (n = 3 separate experiments); ns, not significant. using fluorescence microscopy. Cells were harvested for flow cytometry. Each experiment was independently repeated three times. Visualization of Dendrimer Internalization in eGFP-Expressing Cells. Cells were seeded on 0.1% gelatin-coated glass coverslips. After 24 h cells were incubated with dendrimer (50 μg/mL) /pEGFP (2 μg/mL) complexes for 5 min. Complex-containing medium was replaced with 2.5% (v/v) serum-containing medium and cells incubated for 24 h. The cells were washed with phosphate buffered saline solution (PBS) and fixed with paraformaldehyde 4% (w/v) in PBS for 20 min at room temperature. The cells were rinsed thrice with PBS and permeabilized using PBS containing 0.1% (v/v) Triton X100 during 10 min. After three washes with PBS, cells were incubated with Cy3streptavidin (3:1000 in PBS) for 10 min. Cells were rinsed and mounted on microscopic slides with DAPI-containing mounting medium for confocal fluorescence imaging analysis. Measurement of GFP Expression Using Flow Cytometry. Cells were trypsinized and harvested in 5% (v/v) serum-containing medium, centrifuged, and resuspended in PBS supplemented with 1 μg/mL propidium iodide. Data was collected using BD Accuri C6 flow cytometer for EGFP expression. Analysis of collected data was conducted using FlowJo 10.0.8. All samples were gated using a scatter gate (FSC-A v SSC-A) as to exclude debris, followed by a singlet gate using FSC-A v FSC-W as to exclude doublets. Single cells were then gated for live (PI-negative) cells on a bivariant FL1-A vs FL3-A plot. These live cells were then analyzed for EGFP expression by gating for EGFP expression above the basal fluorescence of control cells using a bivariant EGFP (FL1) v SSC-A plot (Figure S2). Percent (%) transfection efficiency was defined from the proportion of EGFPpositive cells within all live (PI-negative) cells, and the median fluorescent intensity was similarly derived from this EGFP-positive PInegative population. Cell death (%) was defined as proportion of PIpositive cells residing within the single cell gate. Each experiment was separately repeated three times.

produce a biotinylated dendrimer a biotinylated-Lys residue (FmocLys(Biotin)-OH) was introduced between the Gly spacer residue and Lys core (Figure S1). The coupling efficiency was monitored in situ using the colorimetric Ninhydrin test.9 After synthesis of the desired peptide was complete it was cleaved off resin using a mixture of TFA:DCM:H2O (95:2.5:2.5), which was followed by solvent (TFA/ DCM/H2O) removal in vacuo. The resultant residue was then azeotroped with toluene (3 × 15 mL) followed by reconstitution in water, lyophilization and purification using preparative RP-HPLC. Single peak purity was confirmed using analytical RP-HPLC and the target molecular ion [M + H]+ was determined via electron spray ionization mass spectrometry (HR-MS) in positive ion mode. Measurement of Zeta Potential. The net surface charge on dendrimer-DNA complexes was determined via dynamic light scattering phase analysis using a Malvern ZetaSizer Nano ZS. The sample was prepared in 10 mM NaCl, transferred to a zeta cell (Malvern Instruments, DTS1060) and measured at 25 °C with an applied voltage of 150 V. Gel Electrophoretic Assay. Samples were mixed with loading buffer and loaded on a 2% agarose gel containing 10 mg/mL of ethidium bromide. Samples were electrophoresed in 1X TAE buffer (Tris-acetate 10 mM EDTA buffer) at 80 V for 40 min at room temperature, and visualized under UV illumination using an Alphalmager Mini documentation system. Transfection Efficiency Determination. WT, Cav-1 (−/−) and PTRF (−/−) iMEF cells were seeded at a density of 10 000 cells per well in 6-well plates and incubated for 24 h. Cells were then incubated with the dendrimer/DNA complexes (50 μg dendrimer and 1 or 2 μg pEGFP plasmid in a final volume of 1 mL) or with the Lipo/DNA complexes (4 μL of P3000 reagent and 2 μg pEGFP + 3 μL Lipofectamine 3000 in a final volume of 1 mL) for 5 min. In some experiments, the Lipo/DNA complexes were left on the cells for 2.5 h. The GFP expressing live cells were observed and imaged at time points up to 48 h post transfection 440

DOI: 10.1021/acsbiomaterials.6b00033 ACS Biomater. Sci. Eng. 2016, 2, 438−445

Article

ACS Biomaterials Science & Engineering

Figure 3. Comparison of 16+ asymmetric arginine dendrimer pDNA transfection in cells expressing or devoid of caveola-forming proteins. WT, caveolin-1 (−/−) and PTRF (−/−) MEFs were seeded at 10 000 cells per well in 6-well plates and incubated in cell culture medium with 5% serum for 24 h. Cells were exposed for 5 min to either serum-and antibiotic-free medium (control) or to 16+ asymmetric arginine dendrimer (50 μg/mL)/pDNA (2 μg/mL) complex. Transfection mixture was removed and cells were incubated in 2.5% serum (v/v) medium for 48 h and analyzed using flow cytometry. (A) Results are reported as % transfection efficiency and shown as mean ± SEM (n = 6 separate experiments); ns, not significant; *p < 0.05; **p < 0.01. (B) Results are reported as median fluorescence intensity per cell as quantified using FlowJo, and expressed as arbitrary units (AU). Shown are mean ± SEM (n = 6 separate experiments), ns; not significant. (C) Average zeta potential of the biotinylated 16+ asymmetric dendrimer (50 μg/mL), pDNA (2 μg/mL) and 16+ asymmetric dendrimer (50 μg/mL) complexed with pDNA (2 μg/mL) in 10 mM NaCl solution. Results are expressed as mean ± SEM of three separate measurements. Statistical Analysis. All the results are reported as mean ± S.E.M.. Data were analyzed with GraphPad Prism (V6.0 for windows) using one- or two-way ANOVA where appropriate. p values