Article pubs.acs.org/Langmuir
Heterogeneous Cationic Liposomes Modified with 3β‑{N‑[(N′,N′‑Dimethylamino)ethyl]carbamoyl}cholesterol Can Induce Partial Conformational Changes in Messenger RNA and Regulate Translation in an Escherichia coli Cell-Free Translation System Keishi Suga, Tomoyuki Tanabe, and Hiroshi Umakoshi* Division of Chemical Engineering, Graduate School of Engineering Science, Osaka University, 1-3 Machikaneyama-cho, Toyonaka, Osaka 560-8531, Japan S Supporting Information *
ABSTRACT: The effect of cationic liposomes (CLs) on messenger RNA(mRNA) conformation and translation was studied, focusing on membrane heterogeneity. CLs, composed of 1,2-dioleoyl-sn-glycerol-3-phosphocholine/1,2-dioleoyl-3timethylammonium propane (DOPC/DOTAP) and DOPC/ 3β-{N-[(N′,N′-dimethylamino)ethyl]carbamoyl}cholesterol (DOPC/DC-Ch), inhibited mRNA translation in an Escherichia coli cell-free translation system. Analysis of the membrane fluidity and polarity indicated a heterogeneous DOPC/DC-Ch (70/30) membrane, while other CLs exhibited homogeneous disordered membranes. mRNA adsorbed onto DOPC/DC-Ch liposomes showed translational activity, while DOPC/DOTAP liposomes inhibited mRNA translation in proportion to its adsorption onto membranes. Dehydration of DOPC/DOTAP (70/ 30) and DOPC/DC-Ch (70/30) was observed in the presence of mRNA but not in the case of zwitterionic DOPC liposomes, indicating that mRNA binds in regions between the phosphate [-PO2−-] and carbonyl [-C=O-] moieties of lipids. UV resonance Raman spectroscopy suggests that adenine, cytosine, and guanine interact with DOPC/DOTAP (70/30) and DOPC/DC-Ch (70/30) but not with DOPC. Circular dichroism indicates that DOPC/DOTAP (70/30) extensively denatured the mRNA. In contrast, heterogeneous DOPC/DC-Ch (70/30) induced partial conformational changes but maintained the translational activity of mRNA.
■
expression. 2,13 Our previous studies have shown that heterogeneous liposomes containing cholesterol (Ch) significantly enhance biological reactions.14−17 The fundamental mechanism of interaction between liposomes and tRNA14 is as follows: the phosphate group of the tRNA backbone and the trimethylammonium group of lipids interact through electrostatic attraction forces; the nucleobases [adenine (A), uracil (U), cytosine (C), and guanine (G)] and the lipid aliphatic tails interact thorough hydrophobic attraction; and lipid molecules and nucleobases interact via hydrogen bonds. The mechanism of interaction between liposomes and DNA has been described,2,6,7,11,12 but there is little information about interaction between liposomes and RNAs, especially mRNA. Delivery of RNA molecules has a number of advantages in gene therapy strategies: siRNA delivery for RNA interference can knock down target genes, while mRNA can be delivered
INTRODUCTION In recent years, attempts have been made to use gene delivery techniques in various medical and industrial applications (e.g., RNA interference).1 Liposomes, which are assemblies of various kinds of lipids, have been studied as carriers for nucleic acids and drugs, whereby liposome−nucleic acid complexes (lipoplexes) are delivered to target cells through endocytotic or direct membrane fusion pathways.2−4 Modification of liposome surfaces improves the efficiency of gene delivery.4−10 Cationic liposomes (CLs), which are modified with cationic lipids such as 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) and 3β-{N-[(N′,N′-dimethylamino)ethyl]carbamoyl}cholesterol (DC-Ch) (Figure S1, Supporting Information), are quite useful for forming lipoplexes,11,12 although there is little information about the surface properties of CLs. The attractive electrostatic interactions between negatively charged nucleic acids and positively charged CLs containing DOTAP or DC-Ch are very strong. Thus, although CLs can offer higher transfection efficiency in a variety of host cells, these strong electrostatic interactions inhibit the release of the gene, preventing gene © 2013 American Chemical Society
Received: September 9, 2012 Revised: January 13, 2013 Published: January 16, 2013 1899
dx.doi.org/10.1021/la3050576 | Langmuir 2013, 29, 1899−1907
Langmuir
Article
directly to the cytosol and target organelles.18 With regard to synthetic cells, the polynucleotide−lipid membrane interaction has recently attracted significant attention.19−23 One of the essential roles of lipid membranes is to localize functions on the membranes through the binding of biomacromolecules.24 Polynucleotides, such as DNA or RNA, can be functionalized on biomembranes and their mimics, liposomes.25−27 Indeed, lipid membranes can (i) interact with biomacromolecules, (ii) induce minor conformational changes, and thus (iii) regulate their functions. Our previous reports indicate that liposomes regulate in vitro gene expression in an Escherichia coli cell-free translation system,14,16,28−31 depending on the physicochemical properties of the membranes (e.g., surface charge density, membrane fluidity, heterogeneity). Notably, zwitterionic liposomes [1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and POPC/Ch (70/30)] enhanced the expression of green fluorescent protein (GFP), while CLs [DOTAP, POPC/ DOTAP, POPC/stearylamine (SA)] inhibited GFP expression at the translation step. The key step appears to be functionalizing RNAs on the liposome membranes; nevertheless, the translation step is so complicated that the detailed mechanism behind liposome-based interference of translation has not yet been clarified. The aim of this study was to investigate the mechanism behind inhibition of mRNA translation by CLs and to understand the role of the heterogeneous CL in the in vitro gene expression system. Physicochemical properties of CLs were analyzed by use of fluorescent probes: 1,6-diphenyl-1,3,5hexatriene (DPH) for membrane fluidities32,33 and 6-lauroyl-2dimethylaminonaphthalene (Laurdan) for membrane polarities.34−36 Laurdan spectra indicated both an ordered phase (λem 440 nm) and a disordered phase (λem 490 nm) in the DOPC/DC-Ch (70/30) liposome, which suggests that a microscopic phase separation occurs. In the presence of mRNA, dehydration of membrane surfaces was evaluated by variation of general polarity of the Laurdan spectra (general polarization, GP340). The binding sites of mRNA were identified by UV resonance Raman spectroscopy. In addition, we showed that the liposomes affected the conformation of mRNA, which is a key parameter affecting the translational activity of mRNA.37−39 Dependence of the liposome−RNA interaction on the phase state of CLs is also discussed.
■
MLV suspension 11 times through two layers of polycarbonate membrane with mean pore diameter of 100 nm via an extruding device (Liposofast; Avestin Inc., Ottawa, Canada). Liposomes with different compositions were also prepared following the same method. Because liposomes prepared with distilled water and with Tris-HCl buffer solution (100 mM, pH 8.0) provided almost the same results in experiments in the present study, all liposomes were prepared with distilled water. Transcription and Purification of mRNA. pIVEX control vector GFP (Roche) was used as the plasmid DNA. The plasmid DNA was treated once with the restriction enzyme ApaLI for 1 h incubation at 37 °C in order to cleave the AmpR gene and to obtain line DNA fragments harboring the GFP gene before its transcription. Transcription of the mRNA encoding the GFP gene (861 bp) was carried out with T7 RiboMAX expression large-scale RNA production system (Promega, Madison, WI), which includes T7 RNA polymerase as a transcriptional enzyme. Transcription was performed for 30 min at 37 °C. The obtained mRNA was recovered and purified with the SV total RNA isolation kit (Promega, Madison, WI), as previously described.14,28,29 The mRNA products were quantified from absorbance at 258 nm and electrophoresis on 1% agarose gel. Evaluation of mRNA Translational Activity in an E. coli CellFree Translation System. Green fluorescent protein (GFP) was expressed in the cell-free translation system, rapid translation system RTS 100 E. coli HY Kit (RTS kit). The liposomes and mRNA were preincubated for 15 min at 30 °C and then added to the RTS kit. GFP expression was performed for 6 h at 30 °C, and the obtained GFP was kept for 24 h at 4 °C. The amount of GFP synthesized with the RTS kit was evaluated by sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS−PAGE) analysis and the fluorescence of GFP (λex 395 nm, λem 509 nm), on the basis of methods published previously.14,28 Evaluation of Membrane Fluidity of Liposomes. The inner membrane fluidity of the liposomes was evaluated in a similar manner as previous reports.32,33 Fluorescent probe DPH was added to a liposome suspension with a molar ratio of lipid/DPH = 250/1; the final concentrations of lipid and DPH were 100 and 0.4 μM, respectively. The fluorescence polarization of DPH (λex = 360 nm, λem = 430 nm) was measured on a fluorescence spectrophotometer (FP6500 and FP-8500; Jasco, Tokyo, Japan) after incubation at 30 °C for 30 min. The sample was excited with vertically polarized light (360 nm), and emission intensities both perpendicular (I⊥) and parallel (I∥) to the excited light were recorded at 430 nm. The polarization (P) of DPH was then calculated from the following equations:
P = (I − GI⊥)/(I + GI⊥)
G = i⊥/i where i⊥ and i∥ are emission intensity perpendicular and parallel to horizontally polarized light, respectively, and G is the correction factor. The membrane fluidity was evaluated on the basis of the reciprocal of polarization, 1/P. Evaluation of Membrane Polarity of Liposomes by Use of Laurdan. The fluorescent probe Laurdan is sensitive to the polarity around itself, which allows the surface polarity of lipid membranes to be determined.34−36,40,41 Laurdan emission spectra exhibit a red shift caused by dielectric relaxation. Thus, emission spectra were calculated by measuring the general polarization (GP340) for each emission wavelength as follows:
EXPERIMENTAL SECTION
Materials. DOPC, DOTAP, and DC-Ch were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL). Ch was purchased from Sigma−Aldrich (St. Louis, MO). Chemical structures of lipids are shown in Figure S1, Supporting Information. A rapid translation system, RTS 100 E. coli HY kit (RTS kit), was purchased from Roche Diagnostics (Indianapolis, IN). T7 RiboMAX expression large-scale RNA production system and SV total RNA isolation system were purchased from Promega (Madison, WI). Other chemicals were purchased from Wako Pure Chemical (Osaka, Japan) and were used without further purification. Liposome Preparation. A solution of DOPC containing 0−50 mol % DOTAP or DC-Ch, SA in chloroform was dried in a roundbottom flask by rotary evaporation under vacuum. The resulting lipid films were dissolved in chloroform and the solvent evaporated twice. The lipid thin film was kept under high vacuum for at least 3 h and then hydrated with distilled water at room temperature. The vesicle suspension was frozen at −80 °C and then thawed at 50 °C to enhance the transformation of small vesicles into larger multilamellar vesicles (MLVs). This freeze−thaw cycle was repeated five times. MLVs were used to prepare large unilamellar vesicles by extruding the
GP340 = (I440 − I490)/(I440 + I490) where I440 and I490 are the emission intensity of Laurdan excited with 340 nm light. No fluorescence was observed from an mRNA solution (without liposomes). The final concentrations of lipid and Laurdan in the test solution were 100 and 2 μM, respectively. Laurdan spectra were also observed in water/dioxane solutions with different dielectric constant.42 The final concentrations of lipid and Laurdan were 10 and 0.2 μM, respectively. Evaluation of ζ Potential of Cationic Liposomes. Zeta potential of DOPC and CLs was assessed by measuring electro1900
dx.doi.org/10.1021/la3050576 | Langmuir 2013, 29, 1899−1907
Langmuir
Article
phoretic mobility (Zetasizer Nano, Malvern Instruments Ltd., Worcestershire, U.K.).43,44 Final concentration of lipid was 0.5 mM. Agarose Gel Electrophoresis for Evaluation of mRNA Binding onto Cationic Liposomes. mRNA samples were prepared in the presence of DOPC/DOTAP liposomes or DOPC/DC-Ch liposomes, containing 1× loading buffer (0.1% SDS, 5% glycerol, and 0.005% bromophenol blue). The final concentrations of mRNA and lipid were 1 μM and 1 mM, respectively. After 30 min of sample incubation at 30 °C, electrophoresis was performed in a 1% agarose gel with a voltage of 150 mV for 20 min, with 1× TBE running buffer [89 mM Tris, 89 mM boric acid, and 2 mM ethylenediaminetetraacetic acid (EDTA)]. The gel was stained with SYBR Green II, which is an RNA-specific fluorescent probe that enables quantitative analysis of RNA,45 and the density of mRNA bands was analyzed by using the SCION image software obtained at http://www.scion.com/.28 The densitometric analysis was carried out at least three times at different points along the lane. UV Resonance Raman Spectroscopic Analysis. UV resonance Raman spectra of mRNA and nucleoside triphosphates (NTPs) were measured on a confocal Raman microscope (LabRAM HR-800; Horiba, Ltd., Kyoto, Japan) at a wavelength of 266 nm, with laser power of 50 mW and a total data accumulation time of 30 s. For each sample, the background signal of the solution was removed, and then the baseline was corrected. Peak assignments are shown in Supporting Information (Table S1). Peak intensities were normalized to the peak at 878 cm−1 (I878) as an internal reference. The final concentrations of mRNA and lipid in Raman samples were 0.77 μM and 50 or 500 μM, respectively. Evaluation of mRNA Conformation via Circular Dichroic Spectra. The conformation of RNA was evaluated on a Jasco J-820 SFU spectropolarimeter (Jasco, Tokyo).15,16 The circular dichroic (CD) spectrum from 300 to 200 nm was measured with a quartz cell (0.1 cm path length) at a scan speed of 50 nm/min and a width of 2 nm. Five scans excluding buffer and liposome background signals were obtained at 30 °C, and the data were calculated as molar ellipticity. Each sample was prepared with 0.77 μM mRNA and 10 mM Tris-HCl at pH 7.8 in the presence or absence of CLs. Statistical Analysis. Results are expressed as mean ± standard deviation. All experiments were performed at least three times. The distribution of data was analyzed, and statistical differences were evaluated by use of Student’s t-test. A P-value of
