New approaches to improve the traditional gene carriers are still required. Here we explore Fe3O4 modified with degradable polymers that enhances gene delivery and target delivery using permanent magnetic field. Two magnetic Fe3O4 nanoparticles coated with chitosan (CTS) and polyethylene glycol (PEG) were synthesized by means of controlled chemical coprecipitation. Plasmid pEGFP was encapsulated as a reported gene. The ferriferous oxide complexes were approximately spherical; surface charge of CTS-Fe3O4 and PEG-Fe3O4 was about 20 mv and 0 mv, respectively. The controlled release of DNA from the CTS-Fe3O4 nanoparticles was observed. Concurrently, a desired Fe3O4 concentration of less than 2 mM was verified as safe by means of a cytotoxicity test in vitro. Presence of the permanent magnetic field significantly increased the transfection efficiency. Furthermore, the passive target property and safety of magnetic nanoparticles were also demonstrated in an in vivo test. The novel gene delivery system was proved to be an effective tool required for future target expression and gene therapy in vivo.
Nonviral gene vectors have many advantages such as mass production, easier transportation, less immunogenicity, and being easily targeted to organs [
Magnetic ferriferous oxide nanoparticles possess prominent advantages that might correct the defects of traditional drugs and gene carriers. They possess both magnetic and nanoeffects [
The magnetic nanoparticles used as gene carriers are mostly iron oxides. These iron oxides can be generated by precipitation from acidic iron-salt solutions upon addition of appropriate bases [
EGFP was used to monitor gene transfer and gene expression after transfection. The plasmid pEGFP-C1 was propagated in
To observe the target distribution of polymer-Fe3O4 nanoparticles in different organs of mice, 40 pathogen-free BALB/c female mice were purchased from the Sichuan Industrial Institute of Antibiotic for the in vivo studies. The polymer Fe3O4 was redispersed as described previously and injected through the caudal vein on the dosage of 1 mM iron oxide in 0.8 mL. A neodymium-iron-boron (NdFeB) permanent magnet (Br
Release kinetics of plasmid DNA from magnetic nanoparticles were studied [
The polymer-Fe3O4 complexes (1 mM) were mixed with plasmid DNA (4
Human Embryonic Kidney 293 cells (HEK-293), human liver carcinoma cells (HepG2), and mouse myeloma cell line (SP2/0) were maintained in DMEM or RPMI-1640 medium (Gibco-BRL), supplemented with 10% fetal calf serum (FCS, Gibco-BRL) and 1% penicillin/streptomycin. For the transfection and cytotoxicity test, the cells were grown under standard conditions for 24 hours until 70% to 80% confluency in 96-well flat-bottomed microassay plates before the addition of either the plasmid DNA/polymer-Fe3O4 complex or only the polymer Fe3O4.
Assessment of cell viability was performed by the MTT assay. Firstly, the precipitate polymer-Fe3O4 complexes were resuspended under conditions of ultrasonic agitation for 10 min. Subsequently, the complexes were added into the cell-culture fluid at a different concentration (
24-well plates were seeded with 2 × 105 cells (HepG2 and SP2/0 cells) and grown for 24 hours to obtain 70–80% confluence. Prior to transfection, the medium was removed, and the cells were rinsed once with PBS (pH 7.4), then supplied with serum-free medium. The plasmid DNA was mixed with CTS-Fe3O4 and PEG-Fe3O4 as described previously and incubated for 30 minutes at 37°C. DNA/polymer-Fe3O4 complexes were suspended in a serum-free medium to get the final concentrations of 2
TEM images showed that most of the iron oxide complexes were approximately spherical (unpublished data). The XRD measurements also indicated that the samples had a cubic crystal system and magnetite Fe3O4 was the dominant body of the polymer-Fe3O4 complexes. The size and zeta potential showed the two samples to have a uniform size of 100 nm (Figure
The size and zeta potential of the CTS-Fe3O4. (a) Size of distribution of the CTS-Fe3O4; (b) zeta potential of the CTS-Fe3O4.
The different organs from the mice injected with polymer-Fe3O4 were taken out and made into tissue slices. Target distribution of polymer Fe3O4 in vivo was demonstrated with the help of outer static magnetic field. Figure
Target distribution of magnetic CTS-Fe3O4 in liver and lung tissue. Figures were shown by Prussian blue and neutral red staining (×250), with outer static magnetic field for 2 hours. (a) Normal liver tissue; (b) liver tissue injected CTS-Fe3O4 nanoparticles (1 mM); (c) normal lung tissue; (d) lung tissue injected CTS-Fe3O4 nanoparticles (1 mM). Scale bars correspond to 10
Protection of DNA from DNaseI degradation was detected by 1% agarose gel electrophoresis. Naked pEGFP-C1 without digestion and naked pEGFP-C1 following digestion by DNaseI were used as controls. We could evidence partial protection of DNA coated by polymer Fe3O4 from nuclease-mediated DNA degradation (unpublished data). It was assumed that DNA degradation occurs in several layers; external layers will be degraded easily but not internal layers. Furthermore, CTS-Fe3O4 nanoparticles offered higher protection for DNA than PEG-Fe3O4, as the DNA chains could be attached more strongly to the former. In addition, DNaseI digestion resulted in a shift in the most distribution of the DNA isoforms: supercoiled plasmid in nontreated samples was replaced by the open loop form in treated samples.
The in vitro release rates of DNA from polymer-Fe3O4 complexes were studied at different volume ratios. A significant proportion (30%) of the adsorbed DNA was released very rapidly from the CTS-Fe3O4 nanoparticles in the initial 12 hours. After 48 h, the amount of released DNA reached 55% at the optimal E.E. And the remainder of the adsorbed DNA was released slowly, reaching 70% at 96 h (Figure
Kinetics of DNA release from the magnetic nanoparticles in vitro. (a) Percentage of DNA release coated by CTS-Fe3O4 and (b) percentage of DNA release coated by PEG-Fe3O4 at PH 7.4. The data shown are the mean ± standard deviation for three independent experiments.
The N/P ratio (the ratio of negatively charged DNA to positively charged chitosan) is a key factor to determine the optimal complexation conditions. The difference PH and counterions in the medium might directly affect the binding between CTS and DNA [
Low cytotoxicity is one of the major requirements for nonviral vectors for gene delivery. Chitosan was chosen as a functionalizing polysaccharide because of its biocompatibility. It has been reported that chitosan derivatives are less toxic than other cationic polymers such as PEI in vitro and in vivo [
HepG2 and SP2/0 cells were transfected as described previously with either DNA/CTS-Fe3O4 or DNA/PEG-Fe3O4, with DNA/chitosan, DNA/lipofectamine, and naked plasmid as controls. Exposure to a permanent magnetic field (magnet) for 30 min was followed by 4 h incubation. Concurrently, the control groups were routinely transfected using conventional methods. The highest transfection rates were achieved in HepG2 cells corresponding to 67.2% and 45.8% after transfected with CTS-Fe3O4 and PEG-Fe3O4 complexes. Significantly lower transfection rates of 14.3%, 8.7%, and 0.4% resulted from transfection with lipofectamine, chitosan, and naked plasmid, respectively. In addition, the transfection rates were significantly increased by 4.1- and 3.2-fold in HepG2 and SP2/0 cells, when compared to cells not exposed to the magnetic field. Similar transfection results were also obtained with SP2/0 cells, and lower rates of 43.7% and 32.5% treated with CTS-Fe3O4 and PEG-Fe3O4 complexes were achieved. Compared with conventional transfected methods, the results were still statistically significant (Figure
Magnet-assisted transfection of pEGFP plasmid. The SP2/0 cells were transfected with either polymer Fe3O4 or traditional transfection methods in the presence or absence of static magnetic field for 30 min. A and B: magnet-assisted transfection; other groups: traditional transfection. Data are shown as means and SD values from at least three independent experiments (
Magnetic materials modified by biodegradable polymers as gene carriers possess many merits. For examples, simple manufacturing operation, arriving at the target point with the help of an outer magnetic field; a powerful surface energy effect and a small size effect are their outstanding characters. Moreover, it is easy to modify all kinds of multifunctional groups or targeting molecules to form the structure of the core shell, such as CTS, PEI, specific ligands, and monoclonal antibodies, since the complexes have multiple binding sites on their surface, and DNA attaches itself to them in sizeable amounts either through an electrostatic effect or by chemical bond coupling. In order to improve the E.E. of the polymer-Fe3O4 complexes and realize the controlled release of the DNA, we modified the Fe3O4 with multifunctional groups CTS and PEG. In addition, the process of linking polymeric groups did not utilize organic solvent extraction, and the iron content used does not surpass the acceptable daily intake. Furthermore, some of the novel nanoparticles could improve the antigen presentation effect, show a better adjuvant effect, and make a long-term, single-immunization vaccine possible [
CTS-Fe3O4 and PEG-Fe3O4 were successfully prepared. DNA encapsulation efficiency increased with the proportion of polymer-Fe3O4 nanoparticles, and the optimal E.E. (3 : 1) was chosen. Simultaneously, the attachment of DNA to polymer-Fe3O4 complexes did provide protection against cleavage by nuclease. The target distribution of polymer-Fe3O4 complexes with an outer magnetic field was demonstrated in vivo. The controlled-release effect of CTS-Fe3O4 complexes was more apparent than PEG-Fe3O4, and the DNA binding and release from the polymer-Fe3O4 do not alter its functionality. Both CTS-Fe3O4 and PEG-Fe3O4 had low cytotoxicity to HEK-293 and HepG2 cells. The concentration of 2 mM or less in an in vitro application was shown to be absolutely safe. In addition, the magnetic forces lead to an accelerated sedimentation of polymer-Fe3O4 complexes on the cell surface and do directly enhance the transfection efficiency in HepG2 and SP2/0 cells compared with conventional transfection methods. The novel gene delivery system proved to be an effective tool for future, and it is promising in targeting expression and delivery of therapeutic genes in in vivo studies. Our study explored the application of polymer-Fe3O4 nanoparticles as gene carriers. We will continue to do research in this field to provide a more detailed evaluation about the transfer of DNA.
All of the authors have no conflict of interests.
The authors thank the financial support from National Natural Science Foundation of China (Grant no. 30901270).