Biocleavable Polycationic Micelles as Highly Efficient Gene Delivery Vectors
© The Author(s) 2010
Received: 11 April 2010
Accepted: 26 July 2010
Published: 11 August 2010
An amphiphilic disulfide-containing polyamidoamine was synthesized by Michael-type polyaddition reaction of piperazine to equimolar N, N′-bis(acryloyl)cystamine with 90% yield. The polycationic micelles (198 nm, 32.5 mV), prepared from the amphiphilic polyamidoamine by dialysis method, can condense foreign plasmid DNA to form nanosized polycationic micelles/DNA polyelectrolyte complexes with positive charges, which transfected 293T cells with high efficiency. Under optimized conditions, the transfection efficiencies of polycationic micelles/DNA complexes are comparable to, or even higher than that of commercially available branched PEI (Mw 25 kDa).
KeywordsGene delivery Polycationic micelles Biocleavable
Polycationic gene delivery vectors have attracted more and more attention in the past decade because of their versatile chemical structures, ease of preparation, lack of immune response in vivo, etc. [1–4]. Various cationic polymers have been applied in in vitro and in vivo gene delivery, including polylysine (PLL) [5, 6], polyethylenimine (PEI) [7, 8], polyamidoamine dendrimer [9–12], cationic polyester [13, 14], poly(beta-aminoester) [15, 16], polyphosphoester and polyphosphoramidate [17–21]. In general, water-soluble polycations were directly used to condense negatively charged DNA or RNA to from nanosized polyelectrolyte complexes (polyplexes), which can associate with the negatively charged surface of the cells and facilitate the cells uptake of polycation/DNA (or RNA) complexes. Recently, cationic polymeric micelles, prepared from amphiphilic cationic graft or block polymers, were reported as alternative gene delivery vectors [22–31]. The used cationic micelles have core–shell structures composed of hydrophobic segments as core and cationic hydrophilic segments as shell. The cationic polymeric micelles can condense DNA and efficiently deliver foreign DNA into various cell lines. In some cases, the transfection efficiencies of cationic micelles were comparable to polyethylenimine (PEI), even higher than PEI. The significant advantages of cationic polymeric micelles as gene carriers include low cytotoxicity and high transfection efficiency. The usage of polycationic micelles as gene delivery vectors represents the new advances in polymer-based gene delivery. However, most reported cationic polymeric micelles for gene delivery were prepared from block or graft polymer containing hydrophobic polyester segments and hydrophilic polyamine segments. These cationic polymers are often easy to slowly degrade in the process of synthesis and storage, which may limit their practical applications in gene delivery. The goal of this work is to develop novel cationic polymeric micelles for gene delivery, which are stable and nondegradable during the polymer synthesis, preparation and storage of micelles and cationic micelles/DNA complexes. After the uptake of cationic polymeric micelles/DNA complexes by cells, cationic polymers degrade and release DNA under cellular reductive conditions. For this goal, we designed and synthesized an amphiphilic polyamidoamine containing disulfide bonds in the backbone (polymer 1) and investigated the self-assembly of polymer 1 into micelles as efficient gene delivery vectors. Although polymers containing reducible disulfide bonds have been widely applied in biomedical field, such as drug delivery [32–34], gene delivery [35–46] and layer-by-layer assembly [47–49], we here report the first example of the preparation and application of disulfide-containing cationic polymeric micelles in nonviral gene delivery.
Materials and Methods
Cystamine dihydrochloride (97%) was purchased from Fluka and used as received. Dithiothreitol (DTT) was purchased from Merck (Darmstadt, Germany). Other reagents and solvents were of analytical grade obtained from suppliers and used without purification. N,N′-bis(acryloyl) cystamine(BAC) was synthesized from cystamine hydrochloride and acryloyl chloride according to the literature method . FTIR spectra were measured on a Perkin–Elmer Spectrum one spectrometer. 1HNMR spectra were recorded on a Mercury VX-300 MHz instrument. Gel permeation chromatography (GPC) measurement was carried out by using a Waters-2690D HPLC equipped with Shodex K802.5 and K805 columns. Sample was detected with a Wyatt multiangle light scattering detector and a Waters 2,100 differential refractive index detector. Chloroform was used as the mobile phase at a flow rate of 1.0 ml min−1. The green fluorescent protein encoding plasmid pEGFP-C1 was transformed in E. coli DH5α and isolated and purified using Qiagen endotoxin-free plasmid Gigaprep kits according to the supplier’s protocol. The quantity and quality of plasmid DNA was analyzed by spectrophotometric analysis at 260 and 280 nm and by electrophoresis in 0.8% agarose gel. Purified plasmid DNA was diluted in TE buffer and stored at −20°C.
Synthesis and Characterization of Disulfide-Containing Polyamidoamine (Polymer 1)
N,N′-bis(acryloyl)cystamine (BAC) (1.190 g, 4.6 mmol) and piperazine (0.394 g, 4.6 mmol) were mixed in a two-neck flask with a magnetic stirrer under a nitrogen atmosphere. The temperature of the reaction mixture was gradually raised to 100°C and maintained at this temperature for 4 h, and then cooled to room temperature to form slightly yellow solid. The solid was dissolved in 10 ml of methanol and precipitated in a 15-fold excess of acetone. The obtained white powder was dried in vacuum to a constant weight. Yield: 1.432 g (90.4%). FTIR (KBr, cm−1): 3,434, 3,305, 2,940, 2,819, 1,644, 1,548, 1,438, 1,302, 1,131 cm−1. 1H NMR (CDCl3, ppm) : 1.92 (4H, s, CH2CO); 2.45 (4H, t, CH2N); 2.60–2.70 (8H, m, CH2N); 2.85 (4H, t, CH2S); 3.58 (4H, t, CH2); 8.55 (1H, broad, CONH). Mw = 2.17 × 104, Mw/Mn = 1.40.
To a vial, 0.1 ml of pyrene solution in chloroform was added and the solvent was allowed to evaporate to form a thin film at the bottom of the vial. Polymer solutions (5.0 ml) at different concentrations were added to the vials, and the final pyrene concentration was 6 × 10−7 mol l–1 in water. The concentrations of polymer solutions varied from 0.5 to 350 mg l–1. The solutions were kept on a shaker at room temperature for 24 h to reach equilibrium prior to fluorescence runs. The fluorescence spectra were recorded on a Shimadzu RF-5301PC spectrophotometer (Shimadzu, Japan). The emission spectra were scanned from 350 to 450 nm at the excitation wavelength of 333 nm. The slit setting was 7.5 nm. The intensity at 374 nm was analyzed as a function of polymer concentration.
Preparation and Characterization of Disulfide-Containing Polycationic Micelles
Polymer 1 was dissolved in methanol at different concentrations ranging from 0.1 to 5.0 mg ml−1 and then separately dialyzed against ultrapure water using a dialysis tube (MW cut off 8,000–10,000) for 24 h. The water was changed every 4 h. The obtained polymeric micelles solution was directly used for the measurement of size and zeta potential.
Particle Size and Zeta Potential Measurement
The particle size and zeta potential of the freshly prepared polymeric micelles or polymeric micelles/pEGFP-C1 complexes were measured using a Zetasizer (Nano ZS, Malvern Instruments Ltd., UK).
In Vitro Cytotoxicity Assay
The cytotoxicity of disulfide-containing polycationic micelles against 293T cells was evaluated using MTT assay. Before testing, cells in 100 μl of complete DMEM were seeded into 96-well plates at a density of 7,000 cells/well. After 24 h incubation, 100 μl solutions of polycationic micelles in ultrapure water at different concentrations in the range from 0.005 to 2 mg ml−1 were separately added into the wells. The control wells were added with ultrapure water without polycationic micelles. Treated cells were incubated for further 24 h at 37°C. MTT solution (5 mg ml−1, 20 μl) in PBS (0.1 mol l−1, pH 7.4) was added into each well except that PBS (20 μl) was added into the background wells, and the cells were incubated at 37°C for 4 h. After removal of the medium in each well, 150 μl of DMSO was added to each well and the plates was shaken until all the formed formazan blue crystal inside the wells dissolved. The absorbance of the solution in each well at 570 nm was measured using a microplate reader (Bio-Rad 550, Hercules, CA, USA). The percent-relative viability in reference to control wells containing complete DMEM without polymer was calculated by the following equation (A: absorbance at 570 nm, Ablank is the absorbance of the solution containing cells and complete DMEM without MTT and polymer): Relative cell viability (%) = 100 × (Atest−Ablank)/(Acontrol−Ablank).
Preparation of Polymeric Micelles/DNA Complexes
Polymeric micelles/pEGFP-C1 complexes at various N/P ratios were separately prepared by rapidly adding calculated amount of polymeric micelles solution to calculated amount of 150 mmol l−1 sodium chloride containing 1 μg of pEGFP-C1 with gentle vortexing and allowed to incubate at room temperature for half an hour. The total volume of polymeric micelles solution and DNA solution is 100 μl.
Agarose Gel Electrophoresis
Polymeric micelles/DNA complexes at various N/P ratios, ranging from 1 to 40, were separately electrophoresed on 0.7% (W/V) agarose gel containing 0.1% GelRed (V/V) with Tris–acetate–EDTA (TAE) running buffer (pH 8) at 80 V for 60 min. Then, the gel was illuminated on a UV illuminator to show the mobility of DNA. Naked DNA was used as a reference.
Polymeric micelles/pEGFP-C1 complexes at N/P ratio of 20 were selected to study the effect of DTT on the stability of polyplex. DTT solution in 0.15 M NaCl was added to the polyplex solutions to get a final concentration of DTT from 1 to 10 mM, while equal volume of 0.15 M of NaCl without DTT was added to the polyplex solutions as a control. The mixture was incubated at 37°C. At predetermined intervals, the mixture was taken out and electrophoresed on 0.7% (W/V) agarose gel containing 0.1% GelRed (V/V) with Tris–acetate–EDTA (TAE) running buffer (pH 8) at 80 V for 60 min.
In Vitro Transfection and Green Fluorescent Protein Assay
At the density of 5 × 104 cells/well, 293T cells were seeded into a 24-well plate and incubated in 1 ml of complete DMEM for 24 h prior to transfection. The medium was removed and washed gently with PBS (pH 7.4) when the cells were cultured to 40–50% confluency in the 24-well plate. One microliters of serum-free DMEM and 100 μl of 1 μg DNA-containing polymeric micelles/DNA complexes in saline were added into each well. After 4 h incubation at 37°C, the serum-free DMEM was replaced by 1 ml of complete DMEM. After 44 h incubation at 37°C, the cells that expressed green fluorescent proteins were directly observed by inverted fluorescence microscope.
Results and Discussion
Synthesis and Characterization of Disulfide-Containing Polyamidoamine (Polymer 1)
Preparation and Characterization of Polycationic Micelles
Average sizes and zeta potentials of polymeric micelles formed from a solution of polymer 1 in methanol at different concentrations
Concentration (mg ml−1)
Zeta potential (mV)
DNA-binding Ability of Polycationic Micelles
Size and Zeta Potential of Polycationic Micelles/DNA Complexes
Reductive Dissociation of Polymeric Micelles/pEGFP-C1 Complexes and Responsive Release of DNA
In Vitro Transfection of 293T Cells With Polycationic Micelles/DNA Complexes
In summary, an amphiphilic disulfide-containing polyamidoamine homopolymer was synthesized and characterized. The polymer could form cationic micelles with nanosize and moderate positive zeta potential via self-assembly in aqueous solution at low concentration. The formed cationic micelles could condense foreign DNA to form nanosized micelles/DNA complexes and mediate high efficient protein expression in cells at relatively high N/P ratio. Our results indicated that the novel cationic polymeric micelles would be a promising choice in the future nonviral gene delivery.
Min Zhang and Ya-Nan Xue contributed equally to this work.
This work was financially supported by National Natural Science Foundation of China (20874076), National Basic Research Program of China (2005CB623903, 2009CB930300) and Program for New Century Excellent Talents in University (08-0410).
This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.
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