Dalton Transactions PAPER
Cite this: Dalton Trans., 2015, 44, 7058
Tetranuclear ruthenium(II) complexes with oligooxyethylene linkers as one- and two-photon luminescent tracking non-viral gene vectors† Kangqiang Qiu, Bole Yu, Huaiyi Huang, Pingyu Zhang, Liangnian Ji and Hui Chao* To prolong the observation time, increase the penetration depth and decrease self-absorption and phototoxicity, two-photon luminescent vectors have emerged as promising tools for tracking gene delivery in living cells. Herein, we report four new tetranuclear Ru(II) complexes based on oligo-oxyethylene and polybenzimidazole as one- and two- photon luminescent tracking non-viral gene vectors. In such a molecular design, the oligo-oxyethylene, polybenzimidazole and Ru(II) polypyridyl complexes were expected to render the vectors with increased cell permeability, biocompatibility, proton buffering capacity and one- and two-photon luminescence. Corresponding DNA interaction studies showed that
Received 11th January 2015, Accepted 7th March 2015
the ability of the complexes to condense DNA decreased with increasing oligo-oxyethylene lengths.
DOI: 10.1039/c5dt00117j
Additionally, all complexes protected DNA. The complexes were investigated as one- and two-photon tracking non-viral gene vectors in living cells and showed proper cellular uptake, good luciferase
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expression and low cytotoxicity.
1.
Introduction
Gene therapy can be defined as the treatment of human disease by the transfer of genetic material into specific cells of the patient.1,2 Its potential to benefit human health is tremendous, because almost all human diseases have a genetic component, e.g., from intractable genetic disorders to cancer and heart diseases.3–5 Because of several obstacles that must be overcome by the transgene to reach the targeted human cell nuclei, it is necessary to use a gene delivery system that can protect the transgene from degradation and also pass through the plasma membrane to the nucleus.6,7 To date, various synthetic chemical vectors, including lipids,8,9 polymers,10,11 peptides,12 metal complexes3,13–16 and nanomaterials,17,18 have been developed, but few can function as tracking vectors. Ultrasound imaging8 and magnetic resonance imaging19 have been used for tracking in living systems. The most commonly used method for intracellular plasmid trafficking is the fluorescent labelling of vectors with organic dyes,20,21 quantum dots,22,23 carbon dots24 or metal complexes.25 However, this method generally requires multiple complicated
MOE Laboratory of Bioinorganic and Synthetic Chemistry, School of Chemistry and Chemical Engineering, Sun Yat-Sen University, Guangzhou, 510275, P. R. China. E-mail: [email protected]; Tel: +86-20-84112245 † Electronic supplementary information (ESI) available: details of synthesis and characterisation of ligands and Ru(II) complexes, other and supplementary figures. See DOI: 10.1039/c5dt00117j
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reactions. Additionally, the introduction of fluorescent materials may alter the delivery mechanisms and increase potential side-effects.26 Fluorescently labelled trackers have only been used with one-photon microscopy (OPM) and short excitation wavelengths. Most applications of the method do not allow for long-term imaging studies.20–25 For long-term imaging studies, it is crucial to use two-photon microscopy (TPM), which employs two low-energy photons as excitation source to prolong the observation time, increase the penetration depth and decrease self-absorption and phototoxicity.27,28 Ru(II) polypyridyl complexes have emerged as promising luminescent tools for two-photon imaging. These complexes have outstanding photochemical properties, high photostability, large Stokes shifts and high luminescence, and additionally, very good two-photon properties and large two-photon absorption cross sections that make them ideal for tracking studies.29–32 With a high proton buffering capacity, polybenzimidazoles have been used as metal complex ligands for gene delivery.3,14 Our group previously reported a tetranuclear ruthenium(II) complex of polybenzimidazole ligands as a onephoton luminescent non-viral gene vector for use in the realtime tracking of delivery and transfection.33 Polyethylene glycol based polymers, having hydrophilic and lipophilic properties, could increase the vector cell permeability and also provide additional biocompatible properties.34,35 In this work, we report four new tetranuclear ruthenium(II) complexes Ru1–Ru4 (Scheme 1) based on oligo-oxyethylene linkers and
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Scheme 1
Chemical structures of Ru1–Ru4.
polybenzimidazole ligands as one- and two-photon luminescent tracking non-viral gene vectors. After studying the interactions of the complexes with DNA in vitro, the cellular uptake of the Ru-DNA particles was investigated using flow cytometry and transmission electron microscopy (TEM). Then, the complexes were used as one- and two-photon luminescent tracking non-viral gene vectors. The transfection efficiency and cytotoxicity of the DNA condensates were evaluated by luciferase and MTT assays.
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(ESI-MS) were recorded with an LCQ system (Finnigan MAT, USA). Fast atom bombardment mass spectra (FAB-MS) were recorded using a VG ZAB-HS. 1H NMR spectra were recorded with a Varian-500 spectrometer at 25 °C. All chemical shifts are given relative to tetramethylsilane (TMS). UV-visible spectra were recorded with a PerkinElmer Lambda 850 spectrophotometer. Emission spectra were recorded with a PerkinElmer LS 55 spectrofluorophotometer at room temperature (25 °C). Time-resolved emission measurements were conducted with an FLS 920 combined fluorescence-lifetime and steady-state spectrometer. Quantum yields of luminescence at room temperature (25 °C) were calculated according to literature procedures, by using an aerated aqueous solution of [Ru(bpy)3]2+ (ϕ = 0.028) as the reference emitter. All data were processed using the Origin 8 software package. Atomic force microscopy (AFM) images were obtained in air at room temperature with an SPA400 atomic force microscope unit and an SPI3800N control station (Seiko Instruments) operated in tapping mode. Probes were made of a single silicon crystal with a cantilever length of 129 mm and a spring constant of 33–62 N m−1 (OMCLAC160TS-W2, Olympus). Dynamic light scattering and zeta potential experiments were determined by dynamic laser light scattering (DLS) equipment (Brookhaven BI-200SM).
2.3.
Synthesis of the Ru(II) complexes Ru1–Ru4
The ligands (L1–L4) and the complexes (Ru1–Ru4) were synthesized similar to literature methods.38,39 The synthesis and characterizations are described in the ESI.†
2. Experimental section 2.1.
Materials and reagents
All reagents were purchased from commercial sources and used without further purification unless otherwise specified. The plasmid pBR 322 DNA was from MBI Fermentas, the plasmid pEGFP DNA was from Clonetech, and the plasmid pGL3 control vector and luciferase kit were from Promega. Unless otherwise stated, the DNA concentrations are expressed in base pairs. All samples were prepared using distilled water that had been passed through a Millipore-Q ultra-purification system. The compounds 1,2-bis[ phenoxy(3,5-dicarbaldehyde)]ethane, 1,5-bis[ phenoxy(3,5-dicarbaldehyde)]-3-oxopentane, 1,8-bis[ phenoxy(3,5-dicarbaldehyde)]-3,6-dioxooctane, 1,11-bis[ phenoxy(3,5-dicarbaldehyde)]-3,6,9-trioxoundecane,35 1,10phenanthroline-5,6-dione36 and [Ru(bpy)2]Cl2·2H2O37 were prepared according to literature methods. The complexes were dissolved in DMSO prior to the experiments. Then calculated quantities of the complex solutions were added to the appropriate media to yield a final DMSO concentration of less than 1% (v/v). 2.2.
General instrumentation
Microanalyses (C, H and N) were carried out with a vario EL cube elemental analyzer. Electrospray ionization mass spectra
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2.4.
Determination of two-photon absorption cross-sections
The two-photon absorption (TPA) spectra of complexes were determined over a broad spectral region by a two-photon induced luminescence method relative to Rhodamine B in methanol as the standard. The two-photon luminescence data were acquired using an Opolette™ 355II ( pulse width ≤100 fs, 80 MHz repetition rate, tuning range 730–890 nm, Spectra Physics, Inc., USA). Two-photon luminescence measurements were performed in fluorometric quartz cuvettes. The experimental luminescence excitation and detection conditions were such that there were negligible reabsorption processes, which can affect TPA measurements. The quadratic dependence of the two-photon-induced luminescence intensity on the excitation power was verified at excitation wavelengths of 850 nm and 810 nm for Ru1 and Ru2–Ru4, respectively. The TPA crosssection of the probes was calculated at each wavelength according to eqn (1):40 δ2 ¼ δ1
ϕ 1 C 1 I 2 n2 ϕ 2 C 2 I 1 n1
ð1Þ
where I is the integrated luminescence intensity, C is the concentration, n is the refractive index, and ϕ is the quantum yield. The subscript ‘1’ refers to the reference samples and ‘2’ to the experimental samples.
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2.5.
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Complex interaction with DNA
2.5.1. DNA-binding assay. The DNA-binding experiment was performed at room temperature. UV-visible spectra were recorded with a PerkinElmer Lambda 850 spectrophotometer and spectroscopic titrations were carried out in buffer A (5 mM Tris-HCl, 50 mM NaCl, pH = 7.2). The DNA concentration per nucleotide was determined by absorption spectroscopy using the molar absorption coefficient (6600 M−1 cm−1) at 260 nm.41 A solution of calf thymus (CT)-DNA in the buffer gave a ratio of the UV absorbance at 260 nm and 280 nm of 1.8 : 1 to 1.9 : 1, indicating that the DNA was sufficiently free of protein.42 The absorption titration experiments were performed by maintaining a constant Ru(II) complex concentration (5 μM) and by varying the nucleotide concentration (0–110 μM) in buffer. The mixed solutions were allowed to incubate for 5 min before the absorption spectra were recorded. The intrinsic binding constants Kb to DNA were determined using eqn (2):43 ½DNA=ðεa εf Þ ¼ ½DNA=ðεb εf Þ þ 1=K b ðεb εf Þ
ð2Þ
where [DNA] is the concentration of DNA in base pairs, and the apparent absorption coefficients εa, εf, and εb correspond to Aobsd/[Ru], the extinction coefficient for the free ruthenium complex, and the extinction coefficient for the ruthenium complex in the fully bound form, respectively. A plot of [DNA]/ [εa − εf ] versus [DNA] gave a slope of 1/[εa − εf ] and a Y intercept equal to 1/Kb[εb − εf ]. The intrinsic binding constant Kb is given by the ratio of the slope to the intercept. 2.5.2. Preparation of DNA particles. DNA particles were prepared by incubating mixtures containing DNA and Ru(II) complexes at the given +/− ratios in 50 mM Tris-HCl (Tris = tris(hydroxymethyl)aminomethane) solution ( pH = 7.4) or in cell culture, followed by vortexing for 30 min to allow equilibration at room temperature. 2.5.3. Gel retardation assay. Negative supercoiled pBR 322 DNA was treated with Ru(II) complexes in 50 mM Tris-HCl solution ( pH = 7.4), and the solutions were analyzed by electrophoresis for 1.5 h at 75 V on a 1% agarose gel in TBE buffer (89 mM Tris-borate acid, 2 mM EDTA, pH = 8.3). The gel was stained with 1 μg mL−1 ethidium bromide and photographed with an Alpha Innotech IS-5500 fluorescence chemiluminescence and visible imaging system. 2.5.4. Dynamic light scattering and zeta potential assay. DLS equipment was used to determine the average hydrodynamic diameter and the zeta potential of the Ru-pBR 322 DNA particles at various +/− ratios in 50 mM Tris-HCl solution ( pH = 7.4). Typically, 6 runs were measured for each solution, with the average of all runs reported. 2.5.5. AFM imaging. The morphologies of the Ru-pBR 322 DNA particles at the +/− ratios of 53.3, 80.0, 106.7 and 133.3 were examined with AFM. Samples were added dropwise (10 μL) onto a mica substrate, which was freshly cleaved by pulling off the top sheets with tape. One minute later, the substrate was spin-coated (1400 rpm, 30 s) and rinsed with 20 μL of distilled water. AFM images were obtained in air at room temperature. The images were captured in a 256 × 256 pixel
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format and analyzed with the software accompanying the imaging module. 2.5.6. DNase-I protection assay. Ru-pBR 322 DNA particles at the +/− ratios of 53.3, 80.0, 106.7 and 133.3, containing 1 μg pBR 322 DNA and 1 μg free pBR 322 DNA were incubated at 37 °C for 30 min in the presence of 1 unit of DNase-I in digestion buffer consisting of 50 mM Tris-HCl ( pH = 7.4), 2.5 mM MgCl2 and 0.5 mM CaCl2. After DNase-I digestion, the solution was treated with 5 μL of 250 mM EDTA ( pH = 8.0) for 10 min to inactivate DNase-I and then mixed with sodium dodecylsulfate (SDS) in 0.1 M NaOH ( pH = 7.2) at a concentration of 1 wt%. Afterwards, the sample was incubated at room temperature for 2 h and was then run electrophoretically for 1 h using a 1% agarose gel in TBE buffer at 100 V. 2.5.7. DNA photocleavage assay. The photo-induced DNA cleavage by the Ru(II) complexes was examined by gel electrophoresis. Supercoiled pEGFP DNA (0.5 μg) was treated with Ru1–Ru4 at the +/− ratios of 53.3, 80.0, 106.7 and 133.3 in 50 mM Tris-HCl solution ( pH = 7.4). The samples were then irradiated at room temperature with a Xe lamp (450 nm, 150 W). After irradiation, the samples were mixed with SDS at a concentration of 1 wt%. Afterwards, the samples were incubated at room temperature for 2 h and were then run electrophoretically for 1 h using a 1% agarose gel in TBE buffer at 100 V. 2.5.8. Continuous irradiation in presence of DNA. Continuous irradiation in the presence of CT-DNA was performed with a mercury vapor lamp (Osram HBO, 200 W) and a 2000 W quartz halogen lamp (Philips), cooled by water circulation. IR (water) and UV (KNO2) cut-off filters were inserted between the irradiation cell and the exciting source. All experiments were performed with argon- and air-saturated solutions (3 mL) containing Ru(II) complexes (5 μM) and CT-DNA (110 μM, bases). 2.6.
Cell culture conditions and cell viability assay
HeLa cells were maintained as monolayer cultures in DMEM supplemented with 10% fetal bovine serum (FBS) at 37 °C under 5% CO2. Exponentially grown HeLa cells were seeded in triplicate onto 96 well plates at 1 × 104 cells per well. After incubation for 24 h, the cells were treated with increasing concentrations of the tested complexes and Ru-pEGFP DNA particles for 48 h. To stain the viable cells, 20 μL of MTT (5 mg mL−1) was added to each well. The cells were then incubated for 4 h at 37 °C. After the media had been carefully aspirated without disturbing the formed crystals, the dye was dissolved in 200 μL of DMSO. A Tecan Infinite M200 monochromator-based multifunction microplate reader was used to measure the optical density of each well with background subtraction at 590 nm. The cell survival rates in the control wells without Ru(II) complex solutions were considered as 100% cell survival. 2.7.
Cellular uptake of DNA particles
The cells were trypsinized, counted, and adjusted to 1 × 104 cells mL−1 and 1 mL was added to each plate. After 24 h, the cell culture medium was replaced with 800 μL serum-free
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DMEM. Ru-pEGFP DNA particles at the +/− ratios of 53.3, 80.0, 106.7 and 133.3, containing 0.5 μg pEGFP DNA in 200 μL serum-free DMEM were added to the cells and incubated at 37 °C for 4 h. For flow cytometry, after being washed with PBS three times, the cells were trypsinized and centrifuged in PBS buffer. Cells were harvested, and single cell suspensions in 0.5 mL PBS buffer were prepared and subjected to flow cytometry analysis. A flow cytometer (Coulter Co., USA) was used to measure the fluorescence intensity with excitation at 488 nm. For TEM imaging analysis, cell processing was carried out in situ, without displacement from the culture dish. Cells were fixed in a 0.1 M PBS solution containing 2.5% glutaraldehyde and 4% paraformaldehyde for 1 h, rinsed with distilled water, stained with 0.5% uranyl acetate for 1 h, dehydrated in a graded series of ethanol (30, 60, 70, 90 and 100%), and embedded in epoxy resin. The resin was polymerized at 60 °C for 48 h. Ultrathin sections (50–75 nm) obtained with an LKB ultramicrotome were stained with 2% aqueous uranyl acetate and 2% aqueous lead citrate and imaged with a 120 kV FEI Tecnai Spirit TEM. 2.8.
One- and two-photon luminescent imaging
The cells were trypsinized, counted, and adjusted to 1 × 105 cells mL−1, and 1 mL was added to a 35 mm2 Petri dish (MatTek, USA) for laser confocal microscopy. After 24 h, the cell culture medium was replaced with 800 μL serum-free DMEM. Ru-pEGFP DNA particles at the +/− ratios of 53.3, 80.0, 106.7 and 133.3, containing 0.5 μg pEGFP DNA in 200 μL serum-fee DMEM were added to the cells and incubated at 37 °C in the first 4 h. This was followed by a replacement of the medium with fresh DMEM containing 10% FBS, and the cells were incubated for various periods (20 min, and 4, 12 and 24 h). After being washed with fresh PBS ( pH = 7.0) three times, the cells were imaged with a Zeiss LSM 710 NLO confocal microscope (63×/NA 1.4 oil immersion objective). The excitation wavelength of the laser was 488 nm, and the emission spectra were integrated over 580–630 nm (single channel). For two-photon images, the excitation wavelengths of the laser were 850 nm and 810 nm for Ru1 and Ru2–Ru4, respectively. 2.9.
Luciferase assay
HeLa cells were seeded onto a 96 well cell culture plate at a cell density of 1 × 104 cells per well and then incubated for 24 h. Cells were washed with PBS three times and replaced with serum-free DMEM. Ru-plasmid pGL3 control vector particles with increasing concentrations of the tested complexes containing 0.1 μg plasmid were added to the cells and incubated at 37 °C for 4 h. The medium was then replaced with fresh DMEM with 10% FBS and incubated for an additional 24 h. Cells were washed with PBS, harvested and treated for 30 min at 4 °C in an end-over-end rotation with lysis buffer (50 mM Tris-HCl, pH = 7.5, 150 mM NaCl, 2% Triton X-100, 2% NP40). The luciferase assay was carried out according to the manufacturer’s protocol (Promega). Relative light units (RLU) were
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measured with a Varioskan Flash (Thermo Scientific, USA) and a GloMax TM 96 microplate luminometer (Promega, USA).
3. Results and discussion 3.1.
Synthesis and photophysical properties
The synthetic routes for Ru1–Ru4 are shown in Scheme S1.† We started from bis[ phenoxy(3,5-dicarbaldehyde)] (Q1–Q4) which was obtained according to literature methods.35 The ligands (L1–L4) were synthesized with good yield via the condensation of 1,10-phenanthroline-5,6-dione with Q1–Q4. The preparations of the Ru(II) complexes were carried out from the reactions between Ru(bpy)2Cl2 and L1–L4 in ethylene glycol in yields ranging from 73% to 85%.38,39 All these complexes were purified by column chromatography and characterized by elemental analyses, 1H NMR and MS (Fig. S4–S8†). Next, we studied the electronic absorption and emission spectra of Ru1–Ru4 in aqueous media (DMSO–H2O = 1 : 99, v/v) at 298 K. All complexes showed good solubility. The energy maxima and absorption coefficients are summarized in Table S1.† The band at 458–460 nm was assigned to a metal– ligand charge transfer (MLCT) and consisted of overlapping Ru(dπ)→ligand(π*) and Ru(dπ)→bpy(π*) transitions. Excitation into the MLCT band of Ru1–Ru4 at room temperature resulted in a characteristic broad emission peak between 525 nm and 800 nm (Fig. S9†). With a longer linker, the distance between two Ru(II) centers of the complexes was longer and the intensities of the emission spectra of the complexes increased. The emission maxima (Fig. S10†) and relative quantum yields are compiled in Table S1.† A luminescence decay experiment performed at room temperature determined the lifetime of Ru1–Ru4 to be 0.164–0.185 μs through data fitting to a single exponential decay function. The TPA properties of Ru1–Ru4 were also studied. With respect to Rhodamine B,40 the largest TPA cross-sections were 60.0 GM for Ru1, 45.4 GM for Ru2, 32.4 GM for Ru3 and 26.1 GM for Ru4 (1 GM = 1 × 10−50 cm4 s−1·photon−1, Table S1, Fig. S11†). The length of the linker not only influenced the largest TPA cross-sections, but also influenced the largest TPA wavelengths of the complexes. The largest TPA wavelengths were 850 nm for Ru1 and 810 nm for Ru2–Ru4. The twophoton process was confirmed in a power dependence experiment. A log–log linear relationship was observed between the emission intensity and the incident power, with slopes of 2.03 for Ru1, 1.88 for Ru2, 1.96 for Ru3 and 2.07 for Ru4 (Fig. S12†). 3.2.
Interactions with DNA
The first evidence for the ability of Ru1–Ru4 to induce DNA condensation was obtained from an electrophoresis mobility assay performed with plasmid pBR 322 DNA. As shown in Fig. 1, when the concentrations of Ru1–Ru4 increased (varied from 0 to 7 μM), the amount of supercoiled, closed circular pBR 322 DNA (22.5 μM) gradually decreased, and the retardation of DNA in the gel well was increasingly obvious. With a
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Fig. 1 Agarose gel electrophoresis of pBR 322 DNA (22.5 μM) after incubation with Ru1–Ru4 at various +/− ratios in aqueous solution.
Fig. 2 (a) Zeta potential and (b) hydrodynamic diameter of pBR 322 DNA (0.75 μM) incubated with Ru1–Ru4 at various +/− ratios in aqueous solution determined by DLS.
longer linker, the ability of the complexes to condense DNA decreased. This suggested that a concentrated charge was good for condensing DNA. The DNA-binding ability was determined by a DNA (CT-DNA) titration approach. The binding constants were calculated to be 5.18 × 104 M−1, 5.03 × 104 M−1, 4.09 × 104 M−1 and 3.22 × 104 M−1 for Ru1–Ru4 (Fig. S13†). We determined the zeta potential of Ru-pBR 322 DNA particles at various +/− ratios in aqueous solution. As shown in Fig. 2, on the whole, the zeta potential of Ru-DNA particles increased with increasing +/− ratios. For the same +/− ratio, the zeta potential decreased with increasing linker length. The size of Ru-pBR 322 DNA particles at various +/− ratios in aqueous solutions was investigated by DLS. The stable hydrodynamic diameter of Ru-DNA ranged from 200 to 350 nm, and the longer linker particles required higher +/− ratios to be stable. The zeta potential and DLS experiments also indicated that complexes with fewer concentrated charges had less of an ability to condense DNA. To obtain further insight into the DNA particles, we obtained AFM images of the particles at the stable ratios. As shown in Fig. 3, the well-distributed dry DNA particle diameters were similar and ranged from 70 nm to 120 nm. A gene vector should protect pDNA against nucleasecatalyzed biodegradation.44 Therefore, Ru1–Ru4 and pBR 322 DNA at +/− ratios of 53.3, 80.0, 106.7 and 133.3, respectively, were degraded with DNase-I. After the degradation, DNA from the DNA particles migrated approximately the same distance as the control DNA under a fixed electric field (Fig. 4a). This indicated Ru1–Ru4 were able to protect DNA from DNase-I degradation compared with the naked pDNA which did not show any DNA bands after DNase-I digestion.
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Fig. 3 AFM images of pBR 322 DNA (0.75 μM) condensation induced by incubation with Ru1–Ru4 at +/− ratios of 53.3, 80.0, 106.7 and 133.3, respectively.
Fig. 4 (a) pBR 322 DNA protection from DNase-I enzyme by Ru1–Ru4 at +/− ratios of 53.3, 80.0, 106.7 and 133.3, respectively, in the presence of DNase-I enzyme (lanes 1–5). Naked DNA as the control (C); lane 1: naked DNA; lane 2: Ru1; lane 3: Ru2; lane 4: Ru3; lane 5: Ru4. (b) Agarose gel of the photocleavage of pBR 322 DNA with Ru1–Ru4 at +/− ratios of 53.3, 80.0, 106.7 and 133.3 in air, respectively. Naked DNA as the control (C); lane 1: Ru1; lane 2: Ru2; lane 3: Ru3; lane 4: Ru4.
Before the tracking and transfection studies, we performed DNA photodamage experiments with Ru1–Ru4. A DNA photocleavage assay (Fig. 4b) and continuous irradiation experiments (Fig. S14–S15†) showed that Ru1–Ru4 displayed no photocleavage of or photoreactivity towards DNA. 3.3.
Cellular uptake of DNA particles
The prerequisite for an efficient gene transfection is a high DNA uptake level. Flow cytometry was used to quantify the intracellular uptake of Ru-pEGFP DNA particles in HeLa cells. Fig. 5 shows the flow cytometry results of the HeLa cells incubated with Ru-DNA particles and the control untreated cells. Based on the average fluorescence intensity of cells, Ru1 and Ru3 showed an increase in the intracellular delivery of DNA compared with Ru2 and Ru4. We further examined the localization of these DNA particles in HeLa cells by TEM (Fig. 6, S16–S18†). A few Ru1-DNA and
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Fig. 5 Quantitative flow cytometry results of fluorescent intensities of HeLa cells incubated with Ru-pEGFP DNA particles for 4 h. The DNA concentration is 0.75 μM. Ru-DNA particles at +/− ratios of 53.3, 80.0, 106.7 and 133.3, respectively.
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Fig. 7 Time-dependent confocal microscopy images of entry and transportation of Ru1-pEGFP DNA particles at the +/− ratio of 53.3 in HeLa cells. The DNA concentration is 0.75 μM. The red luminescence is Ru1, the blue florescence is Hoechst 33258 and the green florescence is GFP.
detected and higher EGFP expression was found after 24 h for these complexes. Similar behaviors were observed for OPM and TPM excitation. A higher-resolution image was obtained with two-photon technology owing to the two-photon absorption process. 3.5.
Fig. 6 Cellular uptake and intracellular localization of Ru1-pEGFP DNA particles at the +/− ratio of 53.3 monitored by TEM. The DNA concentration is 0.75 μM; N: nucleus, C: cytoplasm.
Luciferase assay
We determined the relative transfection efficiency of this nonviral system by luciferase assays. Plasmid pGL3 was used as a control vector (Fig. 8). As a control, only DNA showed luciferase expression at low levels. The luciferase expression increased when the complexes were used. Ru1 had the highest
Ru3-DNA particles were observed on the cell membrane and more were in the endosome or cytoplasm via endocytosis pathways in HeLa cells. However, Ru2-DNA and Ru4-DNA were only observed in the endosome. The sizes of the DNA particles were similar to the diameters determined by AFM. 3.4.
One- and two-photon imaging
To investigate the intracellular behaviors of Ru-pEGFP DNA particles, one- and two-photon fluorescence microscopy was used to monitor the time-dependent transport and transfection of pEGFP DNA plasmids condensed by these complexes (Fig. 7, S19–S21†). We stained the nuclei of HeLa cells with Hoechst 33258, and then the DNA particles were added to the cells. The Ru-DNA particles were found on the cell membrane in 20 min and more entered the cytoplasm within 4 h. Then, the culture medium was replaced with fresh DMEM containing 10% FBS. After another 12 h, the EGFP expression was
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Fig. 8 Transfection efficiencies of Ru-pGL3 DNA particles in HeLa cells by luciferase assays. The DNA concentration is 0.75 μM. As controls, DNA and lipofectamine 2000 were also investigated.
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luciferase expression. Ru3–Ru4 had similar luciferase expressions. With increasing concentrations of the tested complexes from 10 to 40 μM, the luciferase expression of Ru3 decreased, whereas those of Ru2 and Ru4 increased, and Ru1 had the highest at 20 μM. These observations may be due to the cytotoxicity of the transfection systems.
3.6.
Cell viability
In gene delivery, the cytotoxicity of the vector is major concern. Therefore, we examined the viability of HeLa cells when treated with these transfection systems. As shown in Fig. 9, on the whole, the cytotoxicity of Ru-pEGFP DNA particles was slightly decreased compared with those of Ru1–Ru4. As the concentration increased, the viability of HeLa cells decreased for both the complexes and DNA particles. The cytotoxicity of the Ru3-DNA and Ru4-DNA particles sharply increased as the concentration increased, but the cytotoxicities of the Ru1-DNA and Ru2-DNA particles slowly increased. The cytotoxicity of
these transfection systems was high at high concentrations. At low concentrations, they were relatively safe vectors.
4.
Conclusions
In summary, we synthesized four new tetranuclear ruthenium(II) complexes Ru1–Ru4 based on oligo-oxyethylene linker units, and found that the ability of the complexes to condense DNA decreased with linker length. None of these four complexes damaged DNA. On the contrary, they protected DNA against nuclease-catalyzed biodegradation in vitro. They were used for tracking non-viral gene vectors in living cells due to their good luciferase expressions and low cytotoxicities. Having the highest luciferase expression, Ru1 could have the most potential as a non-viral gene carrier for real-time tracking during delivery and transfection.
Acknowledgements This work was supported by the 973 program (2014CB845604), NSFC (21172273, 21171177, and 91122010), the Program for Changjiang Scholars and the Innovative Research Teams in the University of China (no. IRT1298) and the Research Fund for the Doctoral Program of Higher Education (20110171110013).
Notes and references
Fig. 9 Cytotoxicity of Ru1–Ru4 (a), Ru-pEGFP DNA particles (b) in HeLa cells determined by MTT assays. The DNA concentration is 0.75 μM.
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13 G. Li, R. Guan, L. Ji and H. Chao, Coord. Chem. Rev., 2014, 281, 100–113. 14 J. Yin, X. Meng, S. Zhang, D. Zhang, L. Wang and C. Liu, Biomaterials, 2012, 33, 7884–7894. 15 S. S. Bhat, A. S. Kumbhar, A. A. Kumbhar, A. Khan, P. Lönnecke and E. Hey-Hawkins, Chem. Commun., 2011, 47, 11068–11070. 16 S. S. Bhat, A. S. Kumbha, A. A. Kumbhar and A. Khan, Chem. – Eur. J., 2012, 18, 16383–16392. 17 N. Gandra, D. Wang, Y. Zhu and C. Mao, Angew. Chem., Int. Ed., 2013, 52, 11278–11281. 18 S. B. Hartono, N. T. Phuoc, M. Yu, Z. Jia, M. J. Monteiro, S. Qiao and C. Yu, J. Mater. Chem. B, 2014, 2, 718–726. 19 C. Wu, J. Li, P. Pang, J. Liu, K. Zhu, D. Li, D. Cheng, J. Chen, X. Shuai and H. Shan, Biomaterials, 2014, 35, 8249–8260. 20 R. Savic, L. Luo, A. Eisenberg and D. Maysinger, Science, 2003, 300, 615–618. 21 I. Roy, T. Y. Ohulchanskyy, D. J. Bharali, H. E. Pudavar, R. A. Mistretta, N. Kaur and P. N. Prasad, Proc. Natl. Acad. Sci. U. S. A., 2005, 102, 279–284. 22 J. J. Jung, A. Solanki, K. A. Memoli, K. Kamei, H. Kim, M. A. Drahl, L. J. Williams, H. R. Tseng and K. Lee, Angew. Chem., Int. Ed., 2010, 49, 103–107. 23 Y. Liu, J. Yang, P. Zhang, C. Liu, W. Wang and W. Liu, J. Mater. Chem., 2012, 22, 512–519. 24 J. Kim, J. Park, H. Kim, K. Singha and W. J. Kim, Biomaterials, 2013, 34, 7168–7180. 25 L. Liu, X. Li, S. Hou, Y. Xue, Y. Yao, Y. Ma, X. Feng, S. He, Y. Lu, Y. Wang and X. Zeng, Chem. Commun., 2009, 6759– 6761. 26 U. Resch-Genger, M. Grabolle, S. Cavaliere-Jaricot, R. Nitschke and T. Nann, Nat. Methods, 2008, 5, 763–775. 27 H. Kim and B. Cho, Acc. Chem. Res., 2009, 42, 863–872.
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Cite this: Dalton Trans., 2015, 44, 7058
Tetranuclear ruthenium(II) complexes with oligooxyethylene linkers as one- and two-photon luminescent tracking non-viral gene vectors† Kangqiang Qiu, Bole Yu, Huaiyi Huang, Pingyu Zhang, Liangnian Ji and Hui Chao* To prolong the observation time, increase the penetration depth and decrease self-absorption and phototoxicity, two-photon luminescent vectors have emerged as promising tools for tracking gene delivery in living cells. Herein, we report four new tetranuclear Ru(II) complexes based on oligo-oxyethylene and polybenzimidazole as one- and two- photon luminescent tracking non-viral gene vectors. In such a molecular design, the oligo-oxyethylene, polybenzimidazole and Ru(II) polypyridyl complexes were expected to render the vectors with increased cell permeability, biocompatibility, proton buffering capacity and one- and two-photon luminescence. Corresponding DNA interaction studies showed that
Received 11th January 2015, Accepted 7th March 2015
the ability of the complexes to condense DNA decreased with increasing oligo-oxyethylene lengths.
DOI: 10.1039/c5dt00117j
Additionally, all complexes protected DNA. The complexes were investigated as one- and two-photon tracking non-viral gene vectors in living cells and showed proper cellular uptake, good luciferase
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expression and low cytotoxicity.
1.
Introduction
Gene therapy can be defined as the treatment of human disease by the transfer of genetic material into specific cells of the patient.1,2 Its potential to benefit human health is tremendous, because almost all human diseases have a genetic component, e.g., from intractable genetic disorders to cancer and heart diseases.3–5 Because of several obstacles that must be overcome by the transgene to reach the targeted human cell nuclei, it is necessary to use a gene delivery system that can protect the transgene from degradation and also pass through the plasma membrane to the nucleus.6,7 To date, various synthetic chemical vectors, including lipids,8,9 polymers,10,11 peptides,12 metal complexes3,13–16 and nanomaterials,17,18 have been developed, but few can function as tracking vectors. Ultrasound imaging8 and magnetic resonance imaging19 have been used for tracking in living systems. The most commonly used method for intracellular plasmid trafficking is the fluorescent labelling of vectors with organic dyes,20,21 quantum dots,22,23 carbon dots24 or metal complexes.25 However, this method generally requires multiple complicated
MOE Laboratory of Bioinorganic and Synthetic Chemistry, School of Chemistry and Chemical Engineering, Sun Yat-Sen University, Guangzhou, 510275, P. R. China. E-mail: [email protected]; Tel: +86-20-84112245 † Electronic supplementary information (ESI) available: details of synthesis and characterisation of ligands and Ru(II) complexes, other and supplementary figures. See DOI: 10.1039/c5dt00117j
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reactions. Additionally, the introduction of fluorescent materials may alter the delivery mechanisms and increase potential side-effects.26 Fluorescently labelled trackers have only been used with one-photon microscopy (OPM) and short excitation wavelengths. Most applications of the method do not allow for long-term imaging studies.20–25 For long-term imaging studies, it is crucial to use two-photon microscopy (TPM), which employs two low-energy photons as excitation source to prolong the observation time, increase the penetration depth and decrease self-absorption and phototoxicity.27,28 Ru(II) polypyridyl complexes have emerged as promising luminescent tools for two-photon imaging. These complexes have outstanding photochemical properties, high photostability, large Stokes shifts and high luminescence, and additionally, very good two-photon properties and large two-photon absorption cross sections that make them ideal for tracking studies.29–32 With a high proton buffering capacity, polybenzimidazoles have been used as metal complex ligands for gene delivery.3,14 Our group previously reported a tetranuclear ruthenium(II) complex of polybenzimidazole ligands as a onephoton luminescent non-viral gene vector for use in the realtime tracking of delivery and transfection.33 Polyethylene glycol based polymers, having hydrophilic and lipophilic properties, could increase the vector cell permeability and also provide additional biocompatible properties.34,35 In this work, we report four new tetranuclear ruthenium(II) complexes Ru1–Ru4 (Scheme 1) based on oligo-oxyethylene linkers and
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Dalton Transactions
Scheme 1
Chemical structures of Ru1–Ru4.
polybenzimidazole ligands as one- and two-photon luminescent tracking non-viral gene vectors. After studying the interactions of the complexes with DNA in vitro, the cellular uptake of the Ru-DNA particles was investigated using flow cytometry and transmission electron microscopy (TEM). Then, the complexes were used as one- and two-photon luminescent tracking non-viral gene vectors. The transfection efficiency and cytotoxicity of the DNA condensates were evaluated by luciferase and MTT assays.
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(ESI-MS) were recorded with an LCQ system (Finnigan MAT, USA). Fast atom bombardment mass spectra (FAB-MS) were recorded using a VG ZAB-HS. 1H NMR spectra were recorded with a Varian-500 spectrometer at 25 °C. All chemical shifts are given relative to tetramethylsilane (TMS). UV-visible spectra were recorded with a PerkinElmer Lambda 850 spectrophotometer. Emission spectra were recorded with a PerkinElmer LS 55 spectrofluorophotometer at room temperature (25 °C). Time-resolved emission measurements were conducted with an FLS 920 combined fluorescence-lifetime and steady-state spectrometer. Quantum yields of luminescence at room temperature (25 °C) were calculated according to literature procedures, by using an aerated aqueous solution of [Ru(bpy)3]2+ (ϕ = 0.028) as the reference emitter. All data were processed using the Origin 8 software package. Atomic force microscopy (AFM) images were obtained in air at room temperature with an SPA400 atomic force microscope unit and an SPI3800N control station (Seiko Instruments) operated in tapping mode. Probes were made of a single silicon crystal with a cantilever length of 129 mm and a spring constant of 33–62 N m−1 (OMCLAC160TS-W2, Olympus). Dynamic light scattering and zeta potential experiments were determined by dynamic laser light scattering (DLS) equipment (Brookhaven BI-200SM).
2.3.
Synthesis of the Ru(II) complexes Ru1–Ru4
The ligands (L1–L4) and the complexes (Ru1–Ru4) were synthesized similar to literature methods.38,39 The synthesis and characterizations are described in the ESI.†
2. Experimental section 2.1.
Materials and reagents
All reagents were purchased from commercial sources and used without further purification unless otherwise specified. The plasmid pBR 322 DNA was from MBI Fermentas, the plasmid pEGFP DNA was from Clonetech, and the plasmid pGL3 control vector and luciferase kit were from Promega. Unless otherwise stated, the DNA concentrations are expressed in base pairs. All samples were prepared using distilled water that had been passed through a Millipore-Q ultra-purification system. The compounds 1,2-bis[ phenoxy(3,5-dicarbaldehyde)]ethane, 1,5-bis[ phenoxy(3,5-dicarbaldehyde)]-3-oxopentane, 1,8-bis[ phenoxy(3,5-dicarbaldehyde)]-3,6-dioxooctane, 1,11-bis[ phenoxy(3,5-dicarbaldehyde)]-3,6,9-trioxoundecane,35 1,10phenanthroline-5,6-dione36 and [Ru(bpy)2]Cl2·2H2O37 were prepared according to literature methods. The complexes were dissolved in DMSO prior to the experiments. Then calculated quantities of the complex solutions were added to the appropriate media to yield a final DMSO concentration of less than 1% (v/v). 2.2.
General instrumentation
Microanalyses (C, H and N) were carried out with a vario EL cube elemental analyzer. Electrospray ionization mass spectra
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2.4.
Determination of two-photon absorption cross-sections
The two-photon absorption (TPA) spectra of complexes were determined over a broad spectral region by a two-photon induced luminescence method relative to Rhodamine B in methanol as the standard. The two-photon luminescence data were acquired using an Opolette™ 355II ( pulse width ≤100 fs, 80 MHz repetition rate, tuning range 730–890 nm, Spectra Physics, Inc., USA). Two-photon luminescence measurements were performed in fluorometric quartz cuvettes. The experimental luminescence excitation and detection conditions were such that there were negligible reabsorption processes, which can affect TPA measurements. The quadratic dependence of the two-photon-induced luminescence intensity on the excitation power was verified at excitation wavelengths of 850 nm and 810 nm for Ru1 and Ru2–Ru4, respectively. The TPA crosssection of the probes was calculated at each wavelength according to eqn (1):40 δ2 ¼ δ1
ϕ 1 C 1 I 2 n2 ϕ 2 C 2 I 1 n1
ð1Þ
where I is the integrated luminescence intensity, C is the concentration, n is the refractive index, and ϕ is the quantum yield. The subscript ‘1’ refers to the reference samples and ‘2’ to the experimental samples.
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2.5.
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Complex interaction with DNA
2.5.1. DNA-binding assay. The DNA-binding experiment was performed at room temperature. UV-visible spectra were recorded with a PerkinElmer Lambda 850 spectrophotometer and spectroscopic titrations were carried out in buffer A (5 mM Tris-HCl, 50 mM NaCl, pH = 7.2). The DNA concentration per nucleotide was determined by absorption spectroscopy using the molar absorption coefficient (6600 M−1 cm−1) at 260 nm.41 A solution of calf thymus (CT)-DNA in the buffer gave a ratio of the UV absorbance at 260 nm and 280 nm of 1.8 : 1 to 1.9 : 1, indicating that the DNA was sufficiently free of protein.42 The absorption titration experiments were performed by maintaining a constant Ru(II) complex concentration (5 μM) and by varying the nucleotide concentration (0–110 μM) in buffer. The mixed solutions were allowed to incubate for 5 min before the absorption spectra were recorded. The intrinsic binding constants Kb to DNA were determined using eqn (2):43 ½DNA=ðεa εf Þ ¼ ½DNA=ðεb εf Þ þ 1=K b ðεb εf Þ
ð2Þ
where [DNA] is the concentration of DNA in base pairs, and the apparent absorption coefficients εa, εf, and εb correspond to Aobsd/[Ru], the extinction coefficient for the free ruthenium complex, and the extinction coefficient for the ruthenium complex in the fully bound form, respectively. A plot of [DNA]/ [εa − εf ] versus [DNA] gave a slope of 1/[εa − εf ] and a Y intercept equal to 1/Kb[εb − εf ]. The intrinsic binding constant Kb is given by the ratio of the slope to the intercept. 2.5.2. Preparation of DNA particles. DNA particles were prepared by incubating mixtures containing DNA and Ru(II) complexes at the given +/− ratios in 50 mM Tris-HCl (Tris = tris(hydroxymethyl)aminomethane) solution ( pH = 7.4) or in cell culture, followed by vortexing for 30 min to allow equilibration at room temperature. 2.5.3. Gel retardation assay. Negative supercoiled pBR 322 DNA was treated with Ru(II) complexes in 50 mM Tris-HCl solution ( pH = 7.4), and the solutions were analyzed by electrophoresis for 1.5 h at 75 V on a 1% agarose gel in TBE buffer (89 mM Tris-borate acid, 2 mM EDTA, pH = 8.3). The gel was stained with 1 μg mL−1 ethidium bromide and photographed with an Alpha Innotech IS-5500 fluorescence chemiluminescence and visible imaging system. 2.5.4. Dynamic light scattering and zeta potential assay. DLS equipment was used to determine the average hydrodynamic diameter and the zeta potential of the Ru-pBR 322 DNA particles at various +/− ratios in 50 mM Tris-HCl solution ( pH = 7.4). Typically, 6 runs were measured for each solution, with the average of all runs reported. 2.5.5. AFM imaging. The morphologies of the Ru-pBR 322 DNA particles at the +/− ratios of 53.3, 80.0, 106.7 and 133.3 were examined with AFM. Samples were added dropwise (10 μL) onto a mica substrate, which was freshly cleaved by pulling off the top sheets with tape. One minute later, the substrate was spin-coated (1400 rpm, 30 s) and rinsed with 20 μL of distilled water. AFM images were obtained in air at room temperature. The images were captured in a 256 × 256 pixel
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format and analyzed with the software accompanying the imaging module. 2.5.6. DNase-I protection assay. Ru-pBR 322 DNA particles at the +/− ratios of 53.3, 80.0, 106.7 and 133.3, containing 1 μg pBR 322 DNA and 1 μg free pBR 322 DNA were incubated at 37 °C for 30 min in the presence of 1 unit of DNase-I in digestion buffer consisting of 50 mM Tris-HCl ( pH = 7.4), 2.5 mM MgCl2 and 0.5 mM CaCl2. After DNase-I digestion, the solution was treated with 5 μL of 250 mM EDTA ( pH = 8.0) for 10 min to inactivate DNase-I and then mixed with sodium dodecylsulfate (SDS) in 0.1 M NaOH ( pH = 7.2) at a concentration of 1 wt%. Afterwards, the sample was incubated at room temperature for 2 h and was then run electrophoretically for 1 h using a 1% agarose gel in TBE buffer at 100 V. 2.5.7. DNA photocleavage assay. The photo-induced DNA cleavage by the Ru(II) complexes was examined by gel electrophoresis. Supercoiled pEGFP DNA (0.5 μg) was treated with Ru1–Ru4 at the +/− ratios of 53.3, 80.0, 106.7 and 133.3 in 50 mM Tris-HCl solution ( pH = 7.4). The samples were then irradiated at room temperature with a Xe lamp (450 nm, 150 W). After irradiation, the samples were mixed with SDS at a concentration of 1 wt%. Afterwards, the samples were incubated at room temperature for 2 h and were then run electrophoretically for 1 h using a 1% agarose gel in TBE buffer at 100 V. 2.5.8. Continuous irradiation in presence of DNA. Continuous irradiation in the presence of CT-DNA was performed with a mercury vapor lamp (Osram HBO, 200 W) and a 2000 W quartz halogen lamp (Philips), cooled by water circulation. IR (water) and UV (KNO2) cut-off filters were inserted between the irradiation cell and the exciting source. All experiments were performed with argon- and air-saturated solutions (3 mL) containing Ru(II) complexes (5 μM) and CT-DNA (110 μM, bases). 2.6.
Cell culture conditions and cell viability assay
HeLa cells were maintained as monolayer cultures in DMEM supplemented with 10% fetal bovine serum (FBS) at 37 °C under 5% CO2. Exponentially grown HeLa cells were seeded in triplicate onto 96 well plates at 1 × 104 cells per well. After incubation for 24 h, the cells were treated with increasing concentrations of the tested complexes and Ru-pEGFP DNA particles for 48 h. To stain the viable cells, 20 μL of MTT (5 mg mL−1) was added to each well. The cells were then incubated for 4 h at 37 °C. After the media had been carefully aspirated without disturbing the formed crystals, the dye was dissolved in 200 μL of DMSO. A Tecan Infinite M200 monochromator-based multifunction microplate reader was used to measure the optical density of each well with background subtraction at 590 nm. The cell survival rates in the control wells without Ru(II) complex solutions were considered as 100% cell survival. 2.7.
Cellular uptake of DNA particles
The cells were trypsinized, counted, and adjusted to 1 × 104 cells mL−1 and 1 mL was added to each plate. After 24 h, the cell culture medium was replaced with 800 μL serum-free
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Dalton Transactions
DMEM. Ru-pEGFP DNA particles at the +/− ratios of 53.3, 80.0, 106.7 and 133.3, containing 0.5 μg pEGFP DNA in 200 μL serum-free DMEM were added to the cells and incubated at 37 °C for 4 h. For flow cytometry, after being washed with PBS three times, the cells were trypsinized and centrifuged in PBS buffer. Cells were harvested, and single cell suspensions in 0.5 mL PBS buffer were prepared and subjected to flow cytometry analysis. A flow cytometer (Coulter Co., USA) was used to measure the fluorescence intensity with excitation at 488 nm. For TEM imaging analysis, cell processing was carried out in situ, without displacement from the culture dish. Cells were fixed in a 0.1 M PBS solution containing 2.5% glutaraldehyde and 4% paraformaldehyde for 1 h, rinsed with distilled water, stained with 0.5% uranyl acetate for 1 h, dehydrated in a graded series of ethanol (30, 60, 70, 90 and 100%), and embedded in epoxy resin. The resin was polymerized at 60 °C for 48 h. Ultrathin sections (50–75 nm) obtained with an LKB ultramicrotome were stained with 2% aqueous uranyl acetate and 2% aqueous lead citrate and imaged with a 120 kV FEI Tecnai Spirit TEM. 2.8.
One- and two-photon luminescent imaging
The cells were trypsinized, counted, and adjusted to 1 × 105 cells mL−1, and 1 mL was added to a 35 mm2 Petri dish (MatTek, USA) for laser confocal microscopy. After 24 h, the cell culture medium was replaced with 800 μL serum-free DMEM. Ru-pEGFP DNA particles at the +/− ratios of 53.3, 80.0, 106.7 and 133.3, containing 0.5 μg pEGFP DNA in 200 μL serum-fee DMEM were added to the cells and incubated at 37 °C in the first 4 h. This was followed by a replacement of the medium with fresh DMEM containing 10% FBS, and the cells were incubated for various periods (20 min, and 4, 12 and 24 h). After being washed with fresh PBS ( pH = 7.0) three times, the cells were imaged with a Zeiss LSM 710 NLO confocal microscope (63×/NA 1.4 oil immersion objective). The excitation wavelength of the laser was 488 nm, and the emission spectra were integrated over 580–630 nm (single channel). For two-photon images, the excitation wavelengths of the laser were 850 nm and 810 nm for Ru1 and Ru2–Ru4, respectively. 2.9.
Luciferase assay
HeLa cells were seeded onto a 96 well cell culture plate at a cell density of 1 × 104 cells per well and then incubated for 24 h. Cells were washed with PBS three times and replaced with serum-free DMEM. Ru-plasmid pGL3 control vector particles with increasing concentrations of the tested complexes containing 0.1 μg plasmid were added to the cells and incubated at 37 °C for 4 h. The medium was then replaced with fresh DMEM with 10% FBS and incubated for an additional 24 h. Cells were washed with PBS, harvested and treated for 30 min at 4 °C in an end-over-end rotation with lysis buffer (50 mM Tris-HCl, pH = 7.5, 150 mM NaCl, 2% Triton X-100, 2% NP40). The luciferase assay was carried out according to the manufacturer’s protocol (Promega). Relative light units (RLU) were
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measured with a Varioskan Flash (Thermo Scientific, USA) and a GloMax TM 96 microplate luminometer (Promega, USA).
3. Results and discussion 3.1.
Synthesis and photophysical properties
The synthetic routes for Ru1–Ru4 are shown in Scheme S1.† We started from bis[ phenoxy(3,5-dicarbaldehyde)] (Q1–Q4) which was obtained according to literature methods.35 The ligands (L1–L4) were synthesized with good yield via the condensation of 1,10-phenanthroline-5,6-dione with Q1–Q4. The preparations of the Ru(II) complexes were carried out from the reactions between Ru(bpy)2Cl2 and L1–L4 in ethylene glycol in yields ranging from 73% to 85%.38,39 All these complexes were purified by column chromatography and characterized by elemental analyses, 1H NMR and MS (Fig. S4–S8†). Next, we studied the electronic absorption and emission spectra of Ru1–Ru4 in aqueous media (DMSO–H2O = 1 : 99, v/v) at 298 K. All complexes showed good solubility. The energy maxima and absorption coefficients are summarized in Table S1.† The band at 458–460 nm was assigned to a metal– ligand charge transfer (MLCT) and consisted of overlapping Ru(dπ)→ligand(π*) and Ru(dπ)→bpy(π*) transitions. Excitation into the MLCT band of Ru1–Ru4 at room temperature resulted in a characteristic broad emission peak between 525 nm and 800 nm (Fig. S9†). With a longer linker, the distance between two Ru(II) centers of the complexes was longer and the intensities of the emission spectra of the complexes increased. The emission maxima (Fig. S10†) and relative quantum yields are compiled in Table S1.† A luminescence decay experiment performed at room temperature determined the lifetime of Ru1–Ru4 to be 0.164–0.185 μs through data fitting to a single exponential decay function. The TPA properties of Ru1–Ru4 were also studied. With respect to Rhodamine B,40 the largest TPA cross-sections were 60.0 GM for Ru1, 45.4 GM for Ru2, 32.4 GM for Ru3 and 26.1 GM for Ru4 (1 GM = 1 × 10−50 cm4 s−1·photon−1, Table S1, Fig. S11†). The length of the linker not only influenced the largest TPA cross-sections, but also influenced the largest TPA wavelengths of the complexes. The largest TPA wavelengths were 850 nm for Ru1 and 810 nm for Ru2–Ru4. The twophoton process was confirmed in a power dependence experiment. A log–log linear relationship was observed between the emission intensity and the incident power, with slopes of 2.03 for Ru1, 1.88 for Ru2, 1.96 for Ru3 and 2.07 for Ru4 (Fig. S12†). 3.2.
Interactions with DNA
The first evidence for the ability of Ru1–Ru4 to induce DNA condensation was obtained from an electrophoresis mobility assay performed with plasmid pBR 322 DNA. As shown in Fig. 1, when the concentrations of Ru1–Ru4 increased (varied from 0 to 7 μM), the amount of supercoiled, closed circular pBR 322 DNA (22.5 μM) gradually decreased, and the retardation of DNA in the gel well was increasingly obvious. With a
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Dalton Transactions
Fig. 1 Agarose gel electrophoresis of pBR 322 DNA (22.5 μM) after incubation with Ru1–Ru4 at various +/− ratios in aqueous solution.
Fig. 2 (a) Zeta potential and (b) hydrodynamic diameter of pBR 322 DNA (0.75 μM) incubated with Ru1–Ru4 at various +/− ratios in aqueous solution determined by DLS.
longer linker, the ability of the complexes to condense DNA decreased. This suggested that a concentrated charge was good for condensing DNA. The DNA-binding ability was determined by a DNA (CT-DNA) titration approach. The binding constants were calculated to be 5.18 × 104 M−1, 5.03 × 104 M−1, 4.09 × 104 M−1 and 3.22 × 104 M−1 for Ru1–Ru4 (Fig. S13†). We determined the zeta potential of Ru-pBR 322 DNA particles at various +/− ratios in aqueous solution. As shown in Fig. 2, on the whole, the zeta potential of Ru-DNA particles increased with increasing +/− ratios. For the same +/− ratio, the zeta potential decreased with increasing linker length. The size of Ru-pBR 322 DNA particles at various +/− ratios in aqueous solutions was investigated by DLS. The stable hydrodynamic diameter of Ru-DNA ranged from 200 to 350 nm, and the longer linker particles required higher +/− ratios to be stable. The zeta potential and DLS experiments also indicated that complexes with fewer concentrated charges had less of an ability to condense DNA. To obtain further insight into the DNA particles, we obtained AFM images of the particles at the stable ratios. As shown in Fig. 3, the well-distributed dry DNA particle diameters were similar and ranged from 70 nm to 120 nm. A gene vector should protect pDNA against nucleasecatalyzed biodegradation.44 Therefore, Ru1–Ru4 and pBR 322 DNA at +/− ratios of 53.3, 80.0, 106.7 and 133.3, respectively, were degraded with DNase-I. After the degradation, DNA from the DNA particles migrated approximately the same distance as the control DNA under a fixed electric field (Fig. 4a). This indicated Ru1–Ru4 were able to protect DNA from DNase-I degradation compared with the naked pDNA which did not show any DNA bands after DNase-I digestion.
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Fig. 3 AFM images of pBR 322 DNA (0.75 μM) condensation induced by incubation with Ru1–Ru4 at +/− ratios of 53.3, 80.0, 106.7 and 133.3, respectively.
Fig. 4 (a) pBR 322 DNA protection from DNase-I enzyme by Ru1–Ru4 at +/− ratios of 53.3, 80.0, 106.7 and 133.3, respectively, in the presence of DNase-I enzyme (lanes 1–5). Naked DNA as the control (C); lane 1: naked DNA; lane 2: Ru1; lane 3: Ru2; lane 4: Ru3; lane 5: Ru4. (b) Agarose gel of the photocleavage of pBR 322 DNA with Ru1–Ru4 at +/− ratios of 53.3, 80.0, 106.7 and 133.3 in air, respectively. Naked DNA as the control (C); lane 1: Ru1; lane 2: Ru2; lane 3: Ru3; lane 4: Ru4.
Before the tracking and transfection studies, we performed DNA photodamage experiments with Ru1–Ru4. A DNA photocleavage assay (Fig. 4b) and continuous irradiation experiments (Fig. S14–S15†) showed that Ru1–Ru4 displayed no photocleavage of or photoreactivity towards DNA. 3.3.
Cellular uptake of DNA particles
The prerequisite for an efficient gene transfection is a high DNA uptake level. Flow cytometry was used to quantify the intracellular uptake of Ru-pEGFP DNA particles in HeLa cells. Fig. 5 shows the flow cytometry results of the HeLa cells incubated with Ru-DNA particles and the control untreated cells. Based on the average fluorescence intensity of cells, Ru1 and Ru3 showed an increase in the intracellular delivery of DNA compared with Ru2 and Ru4. We further examined the localization of these DNA particles in HeLa cells by TEM (Fig. 6, S16–S18†). A few Ru1-DNA and
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Dalton Transactions
Fig. 5 Quantitative flow cytometry results of fluorescent intensities of HeLa cells incubated with Ru-pEGFP DNA particles for 4 h. The DNA concentration is 0.75 μM. Ru-DNA particles at +/− ratios of 53.3, 80.0, 106.7 and 133.3, respectively.
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Fig. 7 Time-dependent confocal microscopy images of entry and transportation of Ru1-pEGFP DNA particles at the +/− ratio of 53.3 in HeLa cells. The DNA concentration is 0.75 μM. The red luminescence is Ru1, the blue florescence is Hoechst 33258 and the green florescence is GFP.
detected and higher EGFP expression was found after 24 h for these complexes. Similar behaviors were observed for OPM and TPM excitation. A higher-resolution image was obtained with two-photon technology owing to the two-photon absorption process. 3.5.
Fig. 6 Cellular uptake and intracellular localization of Ru1-pEGFP DNA particles at the +/− ratio of 53.3 monitored by TEM. The DNA concentration is 0.75 μM; N: nucleus, C: cytoplasm.
Luciferase assay
We determined the relative transfection efficiency of this nonviral system by luciferase assays. Plasmid pGL3 was used as a control vector (Fig. 8). As a control, only DNA showed luciferase expression at low levels. The luciferase expression increased when the complexes were used. Ru1 had the highest
Ru3-DNA particles were observed on the cell membrane and more were in the endosome or cytoplasm via endocytosis pathways in HeLa cells. However, Ru2-DNA and Ru4-DNA were only observed in the endosome. The sizes of the DNA particles were similar to the diameters determined by AFM. 3.4.
One- and two-photon imaging
To investigate the intracellular behaviors of Ru-pEGFP DNA particles, one- and two-photon fluorescence microscopy was used to monitor the time-dependent transport and transfection of pEGFP DNA plasmids condensed by these complexes (Fig. 7, S19–S21†). We stained the nuclei of HeLa cells with Hoechst 33258, and then the DNA particles were added to the cells. The Ru-DNA particles were found on the cell membrane in 20 min and more entered the cytoplasm within 4 h. Then, the culture medium was replaced with fresh DMEM containing 10% FBS. After another 12 h, the EGFP expression was
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Fig. 8 Transfection efficiencies of Ru-pGL3 DNA particles in HeLa cells by luciferase assays. The DNA concentration is 0.75 μM. As controls, DNA and lipofectamine 2000 were also investigated.
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luciferase expression. Ru3–Ru4 had similar luciferase expressions. With increasing concentrations of the tested complexes from 10 to 40 μM, the luciferase expression of Ru3 decreased, whereas those of Ru2 and Ru4 increased, and Ru1 had the highest at 20 μM. These observations may be due to the cytotoxicity of the transfection systems.
3.6.
Cell viability
In gene delivery, the cytotoxicity of the vector is major concern. Therefore, we examined the viability of HeLa cells when treated with these transfection systems. As shown in Fig. 9, on the whole, the cytotoxicity of Ru-pEGFP DNA particles was slightly decreased compared with those of Ru1–Ru4. As the concentration increased, the viability of HeLa cells decreased for both the complexes and DNA particles. The cytotoxicity of the Ru3-DNA and Ru4-DNA particles sharply increased as the concentration increased, but the cytotoxicities of the Ru1-DNA and Ru2-DNA particles slowly increased. The cytotoxicity of
these transfection systems was high at high concentrations. At low concentrations, they were relatively safe vectors.
4.
Conclusions
In summary, we synthesized four new tetranuclear ruthenium(II) complexes Ru1–Ru4 based on oligo-oxyethylene linker units, and found that the ability of the complexes to condense DNA decreased with linker length. None of these four complexes damaged DNA. On the contrary, they protected DNA against nuclease-catalyzed biodegradation in vitro. They were used for tracking non-viral gene vectors in living cells due to their good luciferase expressions and low cytotoxicities. Having the highest luciferase expression, Ru1 could have the most potential as a non-viral gene carrier for real-time tracking during delivery and transfection.
Acknowledgements This work was supported by the 973 program (2014CB845604), NSFC (21172273, 21171177, and 91122010), the Program for Changjiang Scholars and the Innovative Research Teams in the University of China (no. IRT1298) and the Research Fund for the Doctoral Program of Higher Education (20110171110013).
Notes and references
Fig. 9 Cytotoxicity of Ru1–Ru4 (a), Ru-pEGFP DNA particles (b) in HeLa cells determined by MTT assays. The DNA concentration is 0.75 μM.
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