Ruthenium-containing supramolecular nanoparticles based on bipyridine-modified cyclodextrin and adamantyl PEI with DNA condensation properties
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Exploring safe and highly efficient gene carriers made from biocompatible constituents has great prospects for clinical gene therapy. Here, a supramolecular gene delivery system was readily constructed by assembling adamantyl-modified polyethylenimine (PEI-Ada) units with a versatile ruthenium bipyridine-modified cyclodextrin (Ru-CD) through host-guest interactions. The photophysical and morphological features of the PEI-Ada@Ru-CD nanoparticles were systematically characterized by techniques including UV-vis absorption spectroscopy, fluorescence spectroscopy, transmission electron microscopy, dynamic light scattering, and zeta potential experiments. The small size and suitably positive zeta potential of the nanoparticles facilitated their cellular uptake and gene transfection. As expected, DNA interaction studies, which were performed using agarose gel electrophoresis and atomic force microscopy, showed that the ability of the nanoparticles to condense DNA was higher than that of the gold standard, i.e., PEI, at low N/P ratios. The design of these ruthenium-containing supramolecular nanoparticles based on bipyridine-modified cyclodextrin and adamantyl PEI has great prospects in the development of gene delivery vehicles.
KeywordsSupramolecular chemistry Self-assembly Ruthenium complexes Cyclodextrin Non-viral gene delivery vector
Atomic force microscopy
β-CD derivative attached carboxylated bipyridines
Dynamic light scattering
Metal-to-ligand charge transfer (dπRu-π*dpb)
Ruthenium bi-pyridine modified cyclodextrin
Transmission electron microscopy
Gene therapy has long been investigated as a promising approach to treat severe diseases [1, 2, 3], such as degenerative diseases, cancer, and genetic diseases. This type of therapy aims to cure diseases by introducing genetic material into cells to alter or replace defective genes [3, 4].
Indeed, for successful gene therapy, an efficient delivery system is required. Viral delivery with a high transfection efficiency was the initial impetus for gene therapy, but the safety of viruses in terms of their toxicity, immunogenicity, and quality scale-up production is a concern . As an alternative, synthetic gene vectors, which have many advantages, including low immunogenicity, a desirable DNA loading capacity, and facile manufacturing, have received much attention . To date, a broad range of non-viral systems for genes, including lipids [7, 8], polymers [9, 10], and peptides [11, 12], have been developed. Polyethylenimine (PEI), a commonly used polycation that electrostatically binds and protects DNA, has emerged as a widely studied non-viral gene vector, as reviewed elsewhere [13, 14, 15]. However, the clinical application of PEI is severely limited by its toxicity; the ED50 of linear PEI was reported to be 4 mg/kg in BALB/C mice . The toxicity of PEI is possibly due to the binding of intracellular and extracellular components at the positively charged surface . Recent studies indicate that the cytotoxicity could be directly reduced by modifying PEI with carbohydrates [17, 18, 19].
Moreover, labelling vectors with organic dyes, quantum dots, carbon dots, or metal complexes has been used for tracking in living systems [20, 21, 22]. Particularly, ruthenium complexes are of considerable interest due to their applications in photochemistry and inorganic pharmacology [23, 24]. Ru(II) polypyridyl complexes have emerged as promising novel agents for cell-staining systems due to their intense luminescence, large Stokes shifts, high chemical and photostability, low energy absorption, and relatively long lifetimes . Additionally, because ruthenium complexes are positively charged transition metal complexes, they can efficiently condense DNA, which is also suitable for gene delivery [25, 26]. For instance, Chao et al. provided a new paradigm for developing non-viral gene vectors for tracking DNA delivery based on a dendritic nanosized hexanuclear Ru(II) polypyridyl complex . Bhat et al. reported the use of two new luminescent ruthenium(II) polypyridyl complexes as carriers for DNA delivery . These studies indicate that ruthenium complexes are attractive candidates for DNA carriers and can be used in the design of multifunctional gene delivery systems. However, gene vectors based on ruthenium complexes usually require multiple complicated reactions.
Supramolecular chemistry is known to be a powerful and convenient approach for constructing gene device systems from individually tunable molecular building blocks [28, 29, 30, 31]. In particular, the construction of supramolecular self-assembly devices based on cyclodextrins (CDs) and their derivatives for use as gene delivery vectors is an active field because of the natural availability, good water solubility, good biocompatibility, and insignificant toxicity of these materials [28, 32, 33].
Results and discussion
Synthesis and complexation of PEI-Ada with Ru-CD
Because the β-CD cavity can strongly bind adamantane derivatives (KS = 104 M−1), the PEI-Ada@Ru-CD supramolecular system could be easily prepared by mixing Ru-CD with PEI-Ada.
Characterization of the PEI-Ada@Ru-CD nanoparticles
The size of PEI-Ada@Ru-CD was also measured by DLS. The particle size of PEI-Ada@Ru-CD was determined to be 263 nm with a normal size distribution. In comparison, the PEI-Ada particles had a diameter of ca. 52 nm. Based on these results, it was concluded that the particle hydrodynamic diameter increased as a function of the number of stabilizing layers after the deposition of PEI-Ada on Ru-CD, whereas the size distribution of the PEI-Ada@Ru-CD nanoclusters might have contributed to the lower degree of aggregation . The hydrodynamic diameter of PEI-Ada@Ru-CD measured by DLS was larger than that calculated from the TEM experiments due to the existence of a hydration layer on the outside of the particles in the aqueous solution and to the shrinkage of the drying nanoparticles during the TEM measurements .
DNA condensation ability
Cell viability and imaging
Zhuo et al. have developed a supramolecular approach for constructing a versatile gene delivery using β-cyclodextrin and adamantyl-functionalized PEIs . Compared with this study, we constructed a ruthenium-containing supramolecular gene deliver. As cationic groups, the PEI head grafted achieved better transfection activity. And β-cyclodextrin might be useful to improve the bioavailability on gene delivery system. More importantly, the β-cyclodextrin and adamantane offered a powerful and convenient method for fabricating complicated nanostructures. More interesting is that the ruthenium-containing nanoparticle not only improves the gene delivery efficiency, but also is a probe for transport imaging.
In conclusion, a supramolecular gene delivery system was readily constructed by assembling adamantyl-modified polyethylenimine (PEI-Ada) units with a versatile Ru-CD through host-guest interactions. The photophysical and morphological features of the PEI-Ada@Ru-CD nanoparticles were systematically characterized by UV-vis absorption spectroscopy, fluorescence spectroscopy, TEM, DLS, and zeta potential experiments. As expected, the DNA interaction studies, which were performed using agarose gel electrophoresis and AFM, showed that the DNA condensation ability of the nanoparticles was higher than that of the gold standard, i.e., PEI, at low N/P ratios. This ruthenium-containing supramolecular gene vector has dual functionalities as a DNA carrier and probe for transport imaging, which opens the door for the further development of novel supramolecular gene delivery systems.
All materials and solvents were purchased from commercial suppliers and used as received unless otherwise stated. 4,4-Dimethyl-2,2-bipyridine, RuCl3·3H2O, dimethyl sulfoxide, branched PEI (MW 25 kDa), β-cyclodextrin, ethidium bromide (EtBr), N-hydroxysuccinimide, and supercoiled pBR322 plasmid DNA (stored at − 20 °C) were purchased from Sigma Chemical Company. Reagent grade β-cyclodextrin was crystallized and recrystallized twice with H2O and dried in vacuo for 24 h at 368 K. Mono[6-(2-aminoethylamino)-6-deoxy]-β-cyclodextrin  and the bipyridine ligands  were synthesized according to literature methods. Adamantane-modified polyethylenimine (PEI-Ada) was synthesized according to a previous report . Purified water was obtained from a Milli-Q Plus (Millipore) system and was used in all experimental solutions.
Methods and instrumentation
UV-vis spectra were recorded at room temperature using a Thermo 300 spectrophotometer (Thermo Electron Corporation, USA). Fluorescence spectroscopic studies were performed using an F-7000 fluorescence spectrophotometer (Hitachi High-Technologies Co., Ltd., Japan).
1H NMR spectra were recorded on a Bruker DMX-400 MHz spectrophotometer with DMSO-d6 as the solvent and SiMe4 (TMS) as the internal standard. High-resolution mass spectra were collected on an LC-MS instrument in the ESI mode. For the DLS measurements, the sample solution was filtered through a 0.80-mm filter into a clean scintillation vial and then examined by a laser light-scattering spectrometer (Nano ZS90, Malvern Instruments, UK) equipped with a digital correlator at 636 nm. AFM images were obtained with a Nanoscope IIIa Multimode AFM (Veeco Company, Multimode, Nano IIIa).
Synthesis of Ru-CD
A solution of the bipyridine-modified cyclodextrin (300 mg, 0.080 mmol) (synthesized according to the literature method shown in Scheme 2, ESI-MS and 1H NMR spectra shown in Additional file 1: Figure. S1–S2) and RuCl3 (5 mg, 0.025 mmol) in 6 mL anhydrous N,N-dimethylformamide was refluxed for 24 h under N2. A small amount of H2O was added, and the mixture was then poured into acetone (150 mL) to give a reddish-brown precipitate. The crude product was obtained by suction filtration, further purified using a Sephadex G-25 column (mobile phase 0.1 M NH3·H2O), and then recrystallized from water/acetone (v/v = 3/1) to give the desired product (220 mg, 65%) as a brown powder: 1H NMR (400 MHz, DMSO-d6, ppm) 9.02 (d, 6H), 7.97 (m, 12H), 5.07–5.30 (m, 42H), 2.99–3.96 (m, 276H).
Preparation of the PEI-Ada@Ru-CD nanoparticles
Briefly, 0.20 mg of the Ru-CD compound and PEI-Ada (24.65 mg, 1.99 mmol) were added to 5 mL distilled water, and the mixture was ultrasonicated for 5 min. Then, the product was collected by centrifugation and washed with water several times. The final solution was stored at 4 °C and diluted to the desired concentration with deionized water before use.
Dynamic light scattering and zeta potential assay
The average hydrodynamic diameter and the zeta potential of the PEI-Ada@Ru-CD nanoparticles were determined by DLS measurements (Nano ZS90, Malvern Instruments, UK). Typically, six runs were performed for each solution, and the average of all the runs was reported.
Transmission electron microscopy (TEM)
The samples were prepared by depositing 5 μL of a PEI-Ada@Ru-CD nanoparticle aqueous solution on a carbon film supported on a 300-mesh copper grid and allowing them to air-dry before collecting the images.
Atomic force microscopy (AFM)
DNA binding experiments were performed by incubating solutions of the PEI-Ada@Ru-CD nanoparticles with pBR322 DNA (2 ng) for approximately 15 min at room temperature. The samples were placed dropwise on a mica substrate, which was freshly cleaved by removing the top layers with tape. The AFM images were obtained on a Nanoscope IIIa Multimode AFM (Veeco Company, Multimode, Nano IIIa) under ambient conditions.
Gel electrophoresis mobility assay
In the gel electrophoresis experiments, negative supercoiled pBR322 DNA was treated with PEI-Ada and PEI-Ada@Ru-CD in 50 mM of a Tris-HCl solution (pH = 7.4), and the mixture was incubated at 37 °C for 30 min. The samples were then analyzed by 1% agarose gel electrophoresis for 1 h at 75 V using a Tris-boric acid-EDTA (TBE) buffer (pH = 8.2) as the running buffer. The gel was stained in a Tris-acetate-EDTA (TAE) buffer containing 5 μg/mL ethidium bromide. The slides were visualized under UV light and photographed for analysis using the Alpha Imager 2200 gel documentation system.
The cytotoxicity of PEI-Ada@Ru-CD/DNA complexes was performed in A549 cells using MTT assay as reported . The A549 cells were seeded into 96-well plates (5000 cells/well) and incubated for 24 h. Thereafter, the complexes with N/P ratios ranging from 5 to 20 were added and the cells were incubated for 48 h, then the fresh medium containing the MTT reagent (0.5 mg/mL) for another 4 h. Finally, the medium was replaced with 150 μL DMSO. The microplate reader (Bio-Rad, Model 550, USA) was used to record the absorbance 570 nm.
The A549 cells were seeded in a 6-well plate. After 24 h, the cell culture medium was changed with 900 μL fresh medium and 100 μL PEI-Ada@Ru-CD/DNA (N/P ratio = 7.2). After being incubated for 8 h, the medium was removed. Subsequently, the cell was fixed with 4% paraformaldehyde before being washed with PBS three times. After that, the Hoechst 33258 was used to stain the nucleus. Then, the cells were imaged using fluorescence microscopy.
We appreciate the experimental assistance of bachelor students Liping Zhang, Yajing Ji, and Yanan Li.
The authors are grateful for the generous financial support of the Project of Shandong Province Higher Educational Science and Technology Program (No. J18KA279), National Natural Science Foundation of China (No. 81774125), Project of Collaborative Innovation Center for Target Drug Delivery System of Weifang Medical University (2017) and College Students’ Technology Innovation Project of Weifang Medical University (KX2017045).
Availability of data and materials
All data are fully available without restriction.
FY performed the experiments and drafted the manuscript. JW and ZL prepared and characterized the PEI-Ada@Ru-CD supramolecular complex. HY and YW performed the statistical design of experiments. WZ and DD proposed the research work. All authors helped to correct and polish the manuscript and read and approved the final manuscript.
The authors declare that they have no competing interests.
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