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Chitosan as a condensing agent induces high gene transfection efficiency and low cytotoxicity of liposome
Journal of Bioscience and Bioengineering VOL. 111 No. 1, 98 – 103, 2011 www.elsevier.com/locate/jbiosc
Chitosan as a condensing agent induces high gene transfection efficiency and low cytotoxicity of liposome Ruizhen Liu, Lu Gan,⁎ Xiangliang Yang, and Huibi Xu College of Life Science and Technology, Huazhong University of Science and Technology, Luoyu Road 1037, Wuhan 430074, China Received 24 March 2010; accepted 23 August 2010 Available online 19 September 2010
To enhance the transfection efficiency of the cationic liposomes, chitosan was selected as a DNA condensing agent. The particle size of the liposome/chitosan/DNA ternary complexes decreased and the zeta potential increased with the addition of chitosan. The formation of the ternary complexes was identified using agarose gel retardation study. The interaction of the ternary complexes was further confirmed by the decrease of the DNA fluorescence in the presence of [Ru(phen)2dppz]2+. In vitro and in vivo transfection activities of the complexes were determined using green fluorescent protein (GFP) expression in various cell lines and mouse tibial anterior muscle subcutaneously, respectively. Liposome/chitosan/DNA ternary complexes showed improved transfection efficiency in vitro cell culture system in the presence or absence of serum as well as in vivo mouse model system, as compared with liposome/DNA lipoplex. More importantly, the cell toxicity of the ternary complex is lower than that of lipoplex and liposome/poly-L-lysine/DNA ternary complex. The precondensation of DNA with chitosan can be a promising approach to further increase the transfection efficiency of cationic liposomes in clinical application. © 2010, The Society for Biotechnology, Japan. All rights reserved. [Key words: Chitosan; Liposome; Gene transfer; Precondensed DNA; Cytotoxicity]
The development of safe, efficient and well-characterized vector systems is one of the greatest limiting factors in gene therapy. Cationic liposomes and cationic polymers are the two major types of non-viral gene delivery vectors currently investigated (1). Because of their cationic charge, both types interact electrostatically with negatively charged DNA and form complexes (lipo- or polyplexes). The cationic DNA complexes obtained were internalized by cells through the electrostatic interaction because of the negatively charged cell membrane. Transfection efficiencies of these complexes have been investigated in vitro and in vivo. However, there are several problems to be overcome before practical use. Those involve insufficient transfection efficiency, strong cytotoxicity, and instability in the serum (2). There have been many reports demonstrating that liposomemediated gene transfer could be augmented by the addition of natural polycations such as protamine sulfate, poly-L-lysine and spermine (3,4). These polycations are known to condense DNA and form a complex with DNA. Although these polycations by themselves mediate DNA delivery, they exhibit a synergistic effect when combined with liposomes in delivering plasmid DNA into several different types of cells. The polycations condense DNA into ternary liposome/polycation/DNA complexes. These particles showed an enhanced gene expression over
⁎ Corresponding author. College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China. Tel.: +86 27 87792147; fax: +86 27 87794517. E-mail address: [email protected] (L. Gan).
that in lipoplex. This enhancement is considered to be caused by a highly compacted complex to assist in an efficient cellular uptake. Chitosan is a naturally occurring polysaccharide consisting of two subunits, D-glucosamine and N-acetyl-D-glucosamine linked together by β (1,4) glycosidic bonds. It was reported as a cationic polymeric condensing carrier for gene delivery (5–7). However, this material has a significant limitation, namely, low transfection efficiency (8). This leads to developing of chitosan nanoparticle system to increase transfection efficiency. In this study, we investigated chitosan as a condensing agent of plasmid DNA to form a ternary complex with liposome and DNA to enhance the transfection activity of liposome. The particle size and zeta potential of the ternary complexes were measured as increasing the ratio of chitosan to DNA. In addition, transmission electron micrographs (TEM) of the ternary complexes were observed. In vitro transfection efficiency was evaluated with the green fluorescent protein (GFP) expression using flow cytometric analysis. The level of in vivo transgene expression was also measured by confocal microscope after injecting the complexes to the mouse tibial anterior muscle. MATERIALS AND METHODS Materials DOTAP (purity N99%) and DOPE (purity N 99%) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). Chitosan (low molecular weight, 50 kD; deacetylation, 90%) was obtained from Fluka (Switzerland). Poly-L-lysine of molecular mass 15–30 kDa was purchased from Sigma (St Louis, MO, USA). Dulbecco's Modified Eagle's medium (DMEM), fetal bovine serum (FBS) were purchased from Gibco BRL (Gaithersberg, MD, USA).
1389-1723/$ - see front matter © 2010, The Society for Biotechnology, Japan. All rights reserved. doi:10.1016/j.jbiosc.2010.08.017
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FIG. 1. Analysis of liposome/DNA binary complexes (A), chitosan/DNA binary complexes (B) and liposome/chitosan/DNA ternary complexes (C) formation by agarose gel electrophoresis. Lane 1: DNA marker. Lane 2: naked DNA. Lanes 3–9 in A: lipoplexes with N/P ratios of 0.5, 1, 1.5, 2, 3, 5 and 10, respectively. Lanes 3–9 in B: chitosan/DNA complexes with N/P ratios of 0.5, 1, 1.5, 2, 3, 5 and 10, respectively. Lanes 3–8 in C: liposome/chitosan/DNA complexes with N/P ratios of 3:0.3:1, 3:0.6:1, 3:0.9:1, 3:1.2:1, 3:1.5:1 and 3:2:1, respectively.
Cell culture Human hepatoma HepG2 cells and mouse fibroblast NIH3T3 cells were purchased from China Center for Type Culture Collection (Wuhan, China). The cells were grown in DMEM medium supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin in the presence of 5% CO2 at 37 °C. The medium was changed twice each weak. Plasmid DNA pVIVO2-GFP plasmid (Invitrogen, San Diego, CA, USA) was used to assess transfection as well as expression efficiency. The plasmid DNA was amplified using Escherichia coli DH5a and purified using the Endo Free Qiagen kit (Qiagen, Valencia, CA, USA) to remove the bacterial endotoxins. The quantity and quality of the purified plasmid DNA were assessed spectrophotometrically at 260 and 280 nm (Shimadzu UV-Biospec 1601, Japan) and also by electrophoresis in agarose gels. Preparation of liposome Cationic liposomes of DOTAP: DOPE were prepared by the film hydration method (9). Briefly, the lipid mixtures containing DOTAP: DOPE (1:1) were dissolved in chloroform. The solvent was then evaporated under vacuum by rotary evaporation to obtain a thin lipid film. The lipid film was maintained under vacuum for at least 30 min to remove any residual solvent, and then hydrated with phosphate-buffered saline (PBS) pH 7.4 for 30 min to achieve a final lipid concentration of 2.5 mM. This preparation was followed by vigorous vortex for 2 min to obtain a solution of multilamellar vesicles. Preparation of chitosan solution Chitosan was dissolved in a 0.3% (v/v) acetic acid by sonication for 30 min. This solution was sterile filtered through a 0.2-μm filter. Preparation of chitosan/liposome/DNA ternary complexes Plasmid DNA was mixed with appropriate amounts of chitosan to obtain the desired N/P ratios. The N/P ratio was based on the calculation of the electrostatic charge present on each component, i.e., the number of terminal NH2 groups in chitosan to the number of phosphate groups in the nucleic acid. After mixing, the solution was briefly vortexed, and the resulting polyplexes were incubated for 20 min at room temperature. The transfection complexes were subsequently prepared by combining each plasmid solution with equal volume of serumfree medium containing cationic liposome. The transfection complexes were incubated for another 20 min at room temperature. Agarose gel electrophoresis After 30-min incubation at room temperature, 4 μl loading dye (6×) were added to 20 μl complexes, and 10 μl of each sample were loaded onto a 0.7% agarose gel. Electrophoresis was carried out at room temperature in TBE buffer at 80 V for 1 h, and ethidium bromide (0.5 μg/ml) was used for DNA staining after gel running according to the standard method. Competition binding assay DNA and [Ru(phen)2dppz]2+ solutions were mixed in TE buffer (10 mM Tris, 1 mM EDTA, pH 7.8) and allowed to equilibrate for 1 min. Appropriate amounts of chitosan with different N/P ratios were added to the solution and incubated for 20 min. Then liposome (the N/P ratio is 3:1) was added to the solution and incubated for another 10 min. The fluorescence was measured on a fluorophotometer with excitation and emission wavelengths of 435 and 598 nm, respectively. Size and zeta-potential measurements The effective hydrodynamic diameter and zeta-potential of the complexes were determined by photon correlation spectroscopy technique using a “ZetaSizer” nanoseries (Malvern Instrument). Transmission electron microscopy (TEM) The size and morphology of liposome/chitosan/DNA complexes prepared at 3:2:1 charge ratio was observed by TEM using carbon coated copper (Cu) grids at 120 kV. One drop of the complex was loaded on the Cu grid for 2 min followed by removing the excess liquid and incubated with 1% uranyl acetate for another 2 min followed by removing the excess liquid. The grid was air-dried for another 2 min and visualized under the electron microscope. Digital images were captured using the instrument software. In vitro transfection Cells were seeded at 5 × 104 cells per well in 24-well dishes and incubated for 24 h at 37 °C under 5% CO2 atmosphere. Immediately before transfection, cells were rinsed and supplemented with 900 μl of fresh culture medium in the presence or absence of FBS. Then, 100 μl of liposome/chitosan/DNA complexes was added in each well and the plates were incubated for 4 h. The transfection complexes were removed, and the cells were rinsed once with serum-containing medium and cultured in
10% FBS-supplemented medium. After 24 h incubation, the transfected cells were harvested and then resuspended in PBS containing 2% formalin. GFP expression levels in the transfected cells were analyzed using a flow cytometer (FACScan, Becton Dickinson, Heidelberg, Germany). The suspended cells were directly introduced to flow cytometer equipped with an argon laser exciting at 488 nm. For each sample, 5000–10000 events were collected by list-mode data, which consisted of side scatter, forward scatter and fluorescence emissions. CellQuest software (Becton Dickinson) was employed to analyze the list-mode data. Determination of GFP positive events was performed by a standard gating technique. Briefly, the control sample was displayed as a dot plot of GFP signals. The gate was drawn along a line of maximum detected GFP intensity for control samples. The percentage of positive events was calculated as the events within the gate divided by total number of events then subtracting percentage of control samples. In vivo transfection Male BALB/c mice (6–8 weeks old) were provided by the Animal Center, Institute of Health and Epidemic Prevention, Wuhan, China. The mice were housed under normal laboratory conditions (21 ± 2 °C, 12/12-h light–dark cycle) with free access to standard rodent chow and water. Comparison of in vivo transfection efficiency was conducted by subcutaneously injecting liposome/chitosan/DNA complex containing the pVIVO2-GFP plasmid (50 μg total DNA), liposome/DNA complex (50 μg total DNA), naked plasmid DNA (50 μg) into the exposed tibialis anterior muscle bundles mice. 2 days later, the muscle was isolated and fixed in 2% paraformaldahyde. The muscle was cut into several segments with the same length and serial crosssections of 7 cm thickness were cut throughout each segment at −20 °C. One out of each 10 sections was selected for examination under confocal microscope and the representative images were reported. Cytotoxicity assays Cytotoxicity of liposome/chitosan/DNA ternary complexes was evaluated by the 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-Diphenyltetrazolium bromide (MTT) assay (10). Cells were seeded at a cell density of 5 × 103 cells/well in 96-well plates and incubated for 24 h. Liposome/DNA lipoplex or liposome/chitosan/DNA ternary complexes were added to the cells in the presence of 10% FBS for 24 h. At the end of the transfection experiments, cells were washed with PBS and then 20 μl of 5 mg/ml MTT solution was added to the cells in each well. Plates were incubated for an additional 2 h at 37 °C. The medium containing MTT was removed and 150 μl of DMSO was added to dissolve the formazan crystals formed by living cells. Absorbance was measured at 490 nm
FIG. 2. Binding behavior of liposome/chitosan/DNA studied by competitive binding studies using [Ru(phen)2dppz]2+. Error bars indicate SD among three individual experiments. Groups 1–7: the N/P ratio of the ternary complexes was 3:0:1, 3:0.3:1, 3:0.6:1, 3:0.9:1, 3:1.2:1, 3:1.5:1 and 3:2:1, respectively.
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FIG. 3. Effect of chitosan on the particle size (A) and zeta potential (B) of liposome/ chitosan/DNA complexes. Complexes were prepared with 50 μg/ml DNA in 10 mM Tris buffer, pH 8.0. Error bars indicate SD among three individual experiments. Groups 1–5: the N/P ratio of the ternary complexes was 3:0:1, 3:0.3:1, 3:0.9:1, 3:1.5:1 and 3:2:1, respectively. Group 6: the N/P ratio of the liposome/polylysine/DNA complex was 3:2:1. *P b 0.05, **P b 0.01 compared with group 1.
using a Labsystems iEMS microplate reader. Results are expressed as the percentage viability with respect to control cell cultures with no complexes added. Statistics Experiments were carried out with three or four replicates. Statistical analyses were performed by Student's t test. Values with P b 0.05 are considered significant.
RESULTS Electrophoresis of the complexes By using an agarose gel electrophoresis technique, the ability of liposome, chitosan and liposome/chitosan complex to interact with DNA was investigated. Consistent with other group's data, DOTAP: DOPE liposome showed strong ability to couple DNA (Fig. 1A) due to electrostatic interaction between liposome and DNA. The extent of DNA retardation in the
FIG. 5. Transfection efficiency of liposome/chitosan/DNA nanoparticles incubated with HepG2 (A) and NIH3T3 (B) cells in the presence or absence of serum. Liposome/chitosan/ DNA nanoparticles containing 1.5 μg of DNA with different N/P ratios were added to the cells and GFP fluorescence was determined by flow cytometric analysis. Error bars indicate SD among three individual experiments. Groups 1–7: the N/P ratio of the ternary complexes was 3:0:1, 3:0.3:1, 3:0.6:1, 3:0.9:1, 3:1.2:1, 3:1.5:1 and 3:2:1, respectively. Group 8: the N/P ratio of the liposome/poly-L-lysine/DNA complex was 3:2:1.
sample well increased with the increasing ratio of liposome/DNA. The DNA was completely retarded in the well but not migrated into the gel when the liposome/DNA ratio comes to 2:1. As expected, chitosan condenses DNA very well. The migration of DNA is retarded completely when the chitosan/DNA ratio is higher than 1.5 (Fig. 1B). However DNA was completely retarded in the well in the liposome/chitosan/DNA ternary complex and the EtBr fluorescence intensity decreased gradually with the addition of chitosan (Fig. 1C).
FIG. 4. Transmission electron microscopy of liposome/chitosan/DNA complex. Liposome/chitosan/DNA complex was prepared at 3:2:1 charge ratio. B is higher magnification of the corresponding images of A. The internal scales are 200 nm (A) and 20 nm (B) respectively.
VOL. 111, 2011 The reason may be that chitosan condenses DNA and then forms a compact complex with liposome which results in the inability of EtBr to intercalate into the DNA double helix. Competitive binding assay The electrostatic nature of interaction of liposome/chitosan/DNA can be examined by competitive binding studies using [Ru(phen)2dppz]2+ as DNA stain. After combination with DNA, the [Ru(phen)2dppz]2+ binds strongly to DNA by intercalating the large aromatic dipyridophenazine (dppz) ring from the direction of the major groove in between the base pairs and results in a remarkable enhancement in luminescence intensity (11). This combination is reversible and can be replaced by other stronger interaction. As shown in Fig. 2, when the DNA/[Ru(phen)2dppz]2+ complex was mixed with liposome/chitosan, the fluorescence intensity of the solution decreased as a result of competitive binding of liposome/chitosan to DNA. The fluorescence decreased further along with the increase of chitosan. Particle size and zeta potential of liposome/chitosan/DNA complexes Since the size and zeta potential of the complexes are important parameters for in vivo and in vitro transfection efficiency, the size and zeta potential of the liposome/chitosan/DNA ternary
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complexes were determined and compared with those of the corresponding lipoplexes. As shown in Fig. 3, the particle size and zeta-potential of the ternary complexes were dependent on the given chitosan/DNA ratios. Following addition of chitosan, the surface potential of the complexes increased gradually. However, the particle size increased at the initial stage because chitosan was distributed at the interface of DNA and the positive charges were insufficient to condense DNA. With the continuous addition of chitosan, DNA was condensed sufficiently to form compact and stable complexes of about 120 nm. The effect of chitosan on the DNA condensing activity and zeta potential in ternary complex was found similar with that of polyL-lysine, a positive control. Transmission electron microscopy (TEM) In order to assess the size and morphology of the liposome/chitosan/DNA complexes, TEM technique was employed. As shown in Fig. 4, the mean size of the liposome/chitosan/DNA complex at 3:2:1 charge ratio is inferior to 150 nm, which confirms the data characterized by photon correlation spectroscopy. The complex appears spherical with a homogenous distribution of DNA within the particles.
FIG. 6. GFP expression in mouse tibialis anterior muscles. After subcutaneous administration of liposome/chitosan/DNA complexes, the muscle was isolated and cut into 7 segments after 2 days. GFP was observed under confocal microscopy. A showed the representative image. a: naked DNA; b–e: the N/P ratio of the ternary complexes was 3:0:1, 3:0.3:1, 3:0.9:1 and 3:1.5:1, respectively. The internal scale is 300 μm. B: GFP expression was quantified.
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In vitro transfection of liposome/chitosan/DNA complexes Liposome/chitosan/DNA ternary complexes were assessed for their in vitro transfection efficiency by monitoring the transient expression of GFP in HepG2 and NIH3T3 cells. HepG2 and NIH3T3 cells were transfected using ternary complexes with different ratios of chitosan/ DNA and GFP expression was determined by flow cytometry. Consistent with the previous data, poly-L-lysine significantly enhanced the transfection efficiency of liposome in the presence or absence of serum. The overall transfection efficacy of liposome/ chitosan/DNA ternary complexes was also dramatically higher than that of lipoplex. The expression levels are dependent on the ratio of chitosan/DNA in ternary complexes. When the N/P ratio comes to 3:2:1, no significant difference in transfection efficiency between liposome/chitosan/DNA and liposome/poly-L-lysine ternary complex was found (Fig. 5). In vivo transfection To determine whether the vitro results could be compatible with in vivo experiments, we assessed the potential of liposome/chitosan/DNA nanoparticles to transfer genes into the tibial anterior muscle. Two days after injection of liposome/ chitosan/DNA nanoparticles, the expression of GFP was monitored using confocal microscopy. As shown in Fig. 6, naked DNA without carriers shows very low transfection efficiency. The transfection efficiency of liposome/DNA complex exceeded that of naked DNA. Upon increasing chitosan in liposome/chitosan/DNA nanoparticles, the expression of GFP significantly increased. Cell viability For eventual in vivo application, a required characteristic of gene delivery systems is that of low cytotoxicity. The application of cationic liposome and treatment with plasmid DNA has often suffered from serious cytotoxic side effects. To determine whether chitosan affects the toxicity of liposome, the effect of the liposome/chitosan/DNA complexes on cell viability was determined using the MTT assay (Fig. 7). Consistent with previous data (12), there is a decrease in cell viability when HepG2 cells and NIH3T3 cells are incubated with lipoplex. Poly-L-lysine further increases the cell toxicity of liposome. However when the cells were treated with liposome/chitosan/DNA ternary complexes, no cytotoxicity was found in these cells, suggesting that chitosan condensation could reduce the cytotoxicity of liposome. DISCUSSION A number of non-viral vectors have been developed for gene therapy because of their higher safety and lower cost. Liposomes and cationic polymers are two major classes of non-viral gene delivery carriers. Although liposomes formed from cationic phospholipids offer several advantages over viral gene transfer, e.g. low immunogenicity and ease of preparation (13), the success of the liposomal approach is limited due to their serious cytotoxic side effects, low transfection efficiency and instability in the serum. Current efforts to improve the transfection efficiency of cationic liposomes focus on the synthesis of new cationic lipids and on the search for better cationic liposome formulations (14). The addition of a polycation, such as polyethylenimine (PEI), protamine sulfate, poly-L-lysine or spermine, has been shown to enhance liposome-mediated gene transfection. This is thought to occur due to electrostatic interactions between the polycation and DNA, resulting in a charge neutralization of the complex and the formation of a condensed structure. It has been shown that some liposome/DNA complexes showed “spaghetti and meatballs” structures (15), suggesting that the DNA molecules are not well condensed in the complexes and they may exist in an extended conformation covered by lipids. In contrast to liposome/DNA complex, the condensed structure of the liposome/polycation/DNA complexes, due to its diminished size, may be more readily endocytosed by the cells and result in the increased levels of transgene expression. However the polycationic nature of these
FIG. 7. Cytotoxicity of liposome/chitosan/DNA nanoparticles on the HepG2 (A) and NIH3T3 (B) cells. Liposome/chitosan/DNA nanoparticles containing 0.5 μg of DNA with different N/P ratios were added to the cells and the toxicity was determined using the MTT assay. Error bars indicate SD among three individual experiments. Group 1: Control group. Groups 2–8: The N/P ratio of the ternary complexes was 3:0:1, 3:0.3:1, 3:0.6:1, 3:0.9:1, 3:1.2:1, 3:1.5:1 and 3:2:1, respectively. Group 9: The N/P ratio of the liposome/poly-L-lysine/DNA complex was 3:2:1. *P b 0.05 compared with group 1.
polycations (e.g. PEI and poly-L-lysine) appeared to be the main origin of their marked toxicity. The toxicity has severely limited their use as gene delivery vectors in vivo. Chitosan is a cheap, biocompatible, biodegradable and non-toxic cationic polymer that forms polyelectrolyte complexes with DNA and considered to be a good candidate for a gene delivery carrier (7). Although chitosan successfully transfected cells in vitro, the transfection efficiency showed to be much lower than that of other cationic polymer vehicles such as PEI (5). Therefore, the combination of liposome and chitosan might be a promising approach for enhancing transfection efficiency. In this study the ability of chitosan as a DNA condensing agent was already shown in Fig. 3 when the size of transfection complex had decreased due to chitosan-added condensation of plasmid DNA. Significant differences were observed in the transfection efficiency of liposome/chitosan/DNA complexes and the charge ratio of chitosan to DNA affected the transfection efficiency of the complexes. The in vitro and in vivo transfection activities of the resulting liposome/chitosan/ DNA ternary complexes are significantly higher than that of the liposome/DNA complex. One possible reason for different transfection efficiency is the difference in their DNA condensing ability. However we could not rule out the possibility that the increased uptake of the complexes also contributed to the enhanced transfection activity
VOL. 111, 2011 considering the increased positive charge of the complexes with increasing chitosan content of the complexes. In this study poly-Llysine was used as a positive control to compare the effect of chitosan on the transfection efficiency of liposome. We found that chitosan showed a similar DNA condensing activity and induced a high transfection efficiency of liposome to a similar extent with poly-L-lysine. It has been reported that chitosan showed significantly lower toxicity than poly-L-lysine and PEI (16,17). In this study the toxicity of the liposome/DNA binary complex and liposome/chitosan/DNA ternary complexes on NIH3T3 and HepG2 cells was evaluated using MTT assay. Consistent with the previous data, liposome/DNA binary complex showed significant cell toxicity and NIH3T3 cells are more sensitive to the toxicity of the liposome/DNA complex than HepG2 cells. Liposome/ poly-L-lysine/DNA ternary complex was more toxic to the cells than liposome/DNA binary complex. In contrast, the liposome/chitosan/DNA ternary complex did not exhibit any noticeable cell toxicity. This probably suggests that chitosan is either acting as a detoxifying agent to reduce the toxicity of liposome/DNA complex or the reduced complex size may have been attributed to lesser cytotoxic effect (18). In the presence of an appropriate amount of chitosan, successful transfection can be achieved with suboptimal ratios of cationic liposomes. In conclusion, we present the development and characterization of a new strategy to prepare more efficient and safe liposome/chitosan/ DNA complexes as an effort to further improve the performance of cationic liposome. These small-sized liposome/chitosan/DNA ternary complexes represent a new class of nonviral gene delivery vehicles that might be useful in gene therapy. However, further understanding of the detailed mechanism underlying the synergism is necessary to improve the design of future gene transfer carriers. References 1. El-Aneed, A.: An overview of current delivery systems in cancer gene therapy, J. Control. Release, 94, 1–14 (2004). 2. Morille, M., Passirani, C., Vonarbourg, A., Clavreul, A., and Benoit, J.: Progress in developing cationic vectors for non-viral systemic gene therapy against cancer, Biomaterials, 29, 3477–3496 (2008).
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3. Gao, X. and Huang, L.: Potentiation of cationic liposome-mediated gene delivery by polycations, Biochemistry, 35, 1027–1036 (1996). 4. Li, S., Rizzo, M., Bhattacharya, S., and Huang, L.: Characterization of cationic lipid– protamine–DNA (LPD) complexes for intravenous gene delivery, Gene Ther., 5, 930–937 (1998). 5. MacLaughlin, F., Mumper, R., Wang, J., Tagliaferri, J., Gill, L., Hinchcliffe, M., and Rolland, A.: Chitosan and depolymerized chitosan oligomers as condensing carriers for in vivo plasmid delivery, J. Control. Release, 56, 259–272 (1998). 6. Rege, P., Shukla, D., and Block, L.: Chitinosans as tableting excipients for modified release delivery systems, Int. J. Pharm., 181, 49–60 (1999). 7. Lai, W. and Lin, M.: Nucleic acid delivery with chitosan and its derivatives, J. Control. Release, 134, 158–168 (2009). 8. Zhao, Q., Chen, J., Han, M., Liang, W., Tabata, Y., and Gao, J.: Combination of poly (ethylenimine) and chitosan induces high gene transfection and low cytotoxicity, J. Biosci. Bioeng., 105, 65–68 (2008). 9. Mirska, D., Schirmer, K., Funari, S., Langner, A., Dobner, B., and Brezesinski, G.: Biophysical and biochemical properties of a binary lipid mixture for DNA transfection, Colloids Surf. B Biointerfaces, 40, 51–59 (2005). 10. Hanelt, M., Gareis, M., and Kollarczik, B.: Cytotoxicity of mycotoxins evaluated by the MTT-i assay, Mycopathologia, 128, 167–174 (1994). 11. Biver, T., Secco, F., and Venturini, M.: Mechanistic aspects of the interaction of intercalating metal complexes with nucleic acids, Coord. Chem. Rev., 252, 1163–1177 (2008). 12. Resina, S., Prevot, P., and Thierry, A.: Physico-chemical characteristics of lipoplexes influence cell uptake mechanisms and transfection efficacy, PLoS ONE, 4, e6058 (2009). 13. Uddin, S.: Cationic lipids used in non-viral gene delivery systems, Biotech. Mol. Biol. Rev., 2, 058–067 (2007). 14. Kim, T., Chung, H., Kwon, I., Sung, H., Shin, B., and Jeong, S.: Polycations enhance emulsion-mediated in vitro and in vivo transfection, Int. J. Pharm., 295, 35–45 (2005). 15. Sternberg, B., Sorgi, F., and Huang, L.: New structures in complex formation between DNA and cationic liposomes visualized by freeze-fracture electron microscopy, FEBS Lett., 356, 361–366 (1994). 16. Erbacher, P., Zou, S., and Remy, J.: Chitosan-based vector/DNA complexes for gene delivery: biophysical characteristics and transfection ability, Pharm. Res., 15, 1332–1339 (1998). 17. Roy, M., Mao, H., and Leong, K.: DNA-chitosan nanospheres: transfection efficacy and cellular uptake, Proc. Int'l. Symp. Control. Rel. Bioact. Mater., 24, 673–674 (1997). 18. Lee, M., Chun, S., Choi, W., Kim, J., Choi, S., Kim, A., Oungbho, K., Park, J., Ahn, W., and Kim, C.: The use of chitosan as a condensing agent to enhance emulsion mediated gene transfer, Biomaterials, 26, 2147–2156 (2005).
Chitosan as a condensing agent induces high gene transfection efficiency and low cytotoxicity of liposome Ruizhen Liu, Lu Gan,⁎ Xiangliang Yang, and Huibi Xu College of Life Science and Technology, Huazhong University of Science and Technology, Luoyu Road 1037, Wuhan 430074, China Received 24 March 2010; accepted 23 August 2010 Available online 19 September 2010
To enhance the transfection efficiency of the cationic liposomes, chitosan was selected as a DNA condensing agent. The particle size of the liposome/chitosan/DNA ternary complexes decreased and the zeta potential increased with the addition of chitosan. The formation of the ternary complexes was identified using agarose gel retardation study. The interaction of the ternary complexes was further confirmed by the decrease of the DNA fluorescence in the presence of [Ru(phen)2dppz]2+. In vitro and in vivo transfection activities of the complexes were determined using green fluorescent protein (GFP) expression in various cell lines and mouse tibial anterior muscle subcutaneously, respectively. Liposome/chitosan/DNA ternary complexes showed improved transfection efficiency in vitro cell culture system in the presence or absence of serum as well as in vivo mouse model system, as compared with liposome/DNA lipoplex. More importantly, the cell toxicity of the ternary complex is lower than that of lipoplex and liposome/poly-L-lysine/DNA ternary complex. The precondensation of DNA with chitosan can be a promising approach to further increase the transfection efficiency of cationic liposomes in clinical application. © 2010, The Society for Biotechnology, Japan. All rights reserved. [Key words: Chitosan; Liposome; Gene transfer; Precondensed DNA; Cytotoxicity]
The development of safe, efficient and well-characterized vector systems is one of the greatest limiting factors in gene therapy. Cationic liposomes and cationic polymers are the two major types of non-viral gene delivery vectors currently investigated (1). Because of their cationic charge, both types interact electrostatically with negatively charged DNA and form complexes (lipo- or polyplexes). The cationic DNA complexes obtained were internalized by cells through the electrostatic interaction because of the negatively charged cell membrane. Transfection efficiencies of these complexes have been investigated in vitro and in vivo. However, there are several problems to be overcome before practical use. Those involve insufficient transfection efficiency, strong cytotoxicity, and instability in the serum (2). There have been many reports demonstrating that liposomemediated gene transfer could be augmented by the addition of natural polycations such as protamine sulfate, poly-L-lysine and spermine (3,4). These polycations are known to condense DNA and form a complex with DNA. Although these polycations by themselves mediate DNA delivery, they exhibit a synergistic effect when combined with liposomes in delivering plasmid DNA into several different types of cells. The polycations condense DNA into ternary liposome/polycation/DNA complexes. These particles showed an enhanced gene expression over
⁎ Corresponding author. College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China. Tel.: +86 27 87792147; fax: +86 27 87794517. E-mail address: [email protected] (L. Gan).
that in lipoplex. This enhancement is considered to be caused by a highly compacted complex to assist in an efficient cellular uptake. Chitosan is a naturally occurring polysaccharide consisting of two subunits, D-glucosamine and N-acetyl-D-glucosamine linked together by β (1,4) glycosidic bonds. It was reported as a cationic polymeric condensing carrier for gene delivery (5–7). However, this material has a significant limitation, namely, low transfection efficiency (8). This leads to developing of chitosan nanoparticle system to increase transfection efficiency. In this study, we investigated chitosan as a condensing agent of plasmid DNA to form a ternary complex with liposome and DNA to enhance the transfection activity of liposome. The particle size and zeta potential of the ternary complexes were measured as increasing the ratio of chitosan to DNA. In addition, transmission electron micrographs (TEM) of the ternary complexes were observed. In vitro transfection efficiency was evaluated with the green fluorescent protein (GFP) expression using flow cytometric analysis. The level of in vivo transgene expression was also measured by confocal microscope after injecting the complexes to the mouse tibial anterior muscle. MATERIALS AND METHODS Materials DOTAP (purity N99%) and DOPE (purity N 99%) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). Chitosan (low molecular weight, 50 kD; deacetylation, 90%) was obtained from Fluka (Switzerland). Poly-L-lysine of molecular mass 15–30 kDa was purchased from Sigma (St Louis, MO, USA). Dulbecco's Modified Eagle's medium (DMEM), fetal bovine serum (FBS) were purchased from Gibco BRL (Gaithersberg, MD, USA).
1389-1723/$ - see front matter © 2010, The Society for Biotechnology, Japan. All rights reserved. doi:10.1016/j.jbiosc.2010.08.017
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FIG. 1. Analysis of liposome/DNA binary complexes (A), chitosan/DNA binary complexes (B) and liposome/chitosan/DNA ternary complexes (C) formation by agarose gel electrophoresis. Lane 1: DNA marker. Lane 2: naked DNA. Lanes 3–9 in A: lipoplexes with N/P ratios of 0.5, 1, 1.5, 2, 3, 5 and 10, respectively. Lanes 3–9 in B: chitosan/DNA complexes with N/P ratios of 0.5, 1, 1.5, 2, 3, 5 and 10, respectively. Lanes 3–8 in C: liposome/chitosan/DNA complexes with N/P ratios of 3:0.3:1, 3:0.6:1, 3:0.9:1, 3:1.2:1, 3:1.5:1 and 3:2:1, respectively.
Cell culture Human hepatoma HepG2 cells and mouse fibroblast NIH3T3 cells were purchased from China Center for Type Culture Collection (Wuhan, China). The cells were grown in DMEM medium supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin in the presence of 5% CO2 at 37 °C. The medium was changed twice each weak. Plasmid DNA pVIVO2-GFP plasmid (Invitrogen, San Diego, CA, USA) was used to assess transfection as well as expression efficiency. The plasmid DNA was amplified using Escherichia coli DH5a and purified using the Endo Free Qiagen kit (Qiagen, Valencia, CA, USA) to remove the bacterial endotoxins. The quantity and quality of the purified plasmid DNA were assessed spectrophotometrically at 260 and 280 nm (Shimadzu UV-Biospec 1601, Japan) and also by electrophoresis in agarose gels. Preparation of liposome Cationic liposomes of DOTAP: DOPE were prepared by the film hydration method (9). Briefly, the lipid mixtures containing DOTAP: DOPE (1:1) were dissolved in chloroform. The solvent was then evaporated under vacuum by rotary evaporation to obtain a thin lipid film. The lipid film was maintained under vacuum for at least 30 min to remove any residual solvent, and then hydrated with phosphate-buffered saline (PBS) pH 7.4 for 30 min to achieve a final lipid concentration of 2.5 mM. This preparation was followed by vigorous vortex for 2 min to obtain a solution of multilamellar vesicles. Preparation of chitosan solution Chitosan was dissolved in a 0.3% (v/v) acetic acid by sonication for 30 min. This solution was sterile filtered through a 0.2-μm filter. Preparation of chitosan/liposome/DNA ternary complexes Plasmid DNA was mixed with appropriate amounts of chitosan to obtain the desired N/P ratios. The N/P ratio was based on the calculation of the electrostatic charge present on each component, i.e., the number of terminal NH2 groups in chitosan to the number of phosphate groups in the nucleic acid. After mixing, the solution was briefly vortexed, and the resulting polyplexes were incubated for 20 min at room temperature. The transfection complexes were subsequently prepared by combining each plasmid solution with equal volume of serumfree medium containing cationic liposome. The transfection complexes were incubated for another 20 min at room temperature. Agarose gel electrophoresis After 30-min incubation at room temperature, 4 μl loading dye (6×) were added to 20 μl complexes, and 10 μl of each sample were loaded onto a 0.7% agarose gel. Electrophoresis was carried out at room temperature in TBE buffer at 80 V for 1 h, and ethidium bromide (0.5 μg/ml) was used for DNA staining after gel running according to the standard method. Competition binding assay DNA and [Ru(phen)2dppz]2+ solutions were mixed in TE buffer (10 mM Tris, 1 mM EDTA, pH 7.8) and allowed to equilibrate for 1 min. Appropriate amounts of chitosan with different N/P ratios were added to the solution and incubated for 20 min. Then liposome (the N/P ratio is 3:1) was added to the solution and incubated for another 10 min. The fluorescence was measured on a fluorophotometer with excitation and emission wavelengths of 435 and 598 nm, respectively. Size and zeta-potential measurements The effective hydrodynamic diameter and zeta-potential of the complexes were determined by photon correlation spectroscopy technique using a “ZetaSizer” nanoseries (Malvern Instrument). Transmission electron microscopy (TEM) The size and morphology of liposome/chitosan/DNA complexes prepared at 3:2:1 charge ratio was observed by TEM using carbon coated copper (Cu) grids at 120 kV. One drop of the complex was loaded on the Cu grid for 2 min followed by removing the excess liquid and incubated with 1% uranyl acetate for another 2 min followed by removing the excess liquid. The grid was air-dried for another 2 min and visualized under the electron microscope. Digital images were captured using the instrument software. In vitro transfection Cells were seeded at 5 × 104 cells per well in 24-well dishes and incubated for 24 h at 37 °C under 5% CO2 atmosphere. Immediately before transfection, cells were rinsed and supplemented with 900 μl of fresh culture medium in the presence or absence of FBS. Then, 100 μl of liposome/chitosan/DNA complexes was added in each well and the plates were incubated for 4 h. The transfection complexes were removed, and the cells were rinsed once with serum-containing medium and cultured in
10% FBS-supplemented medium. After 24 h incubation, the transfected cells were harvested and then resuspended in PBS containing 2% formalin. GFP expression levels in the transfected cells were analyzed using a flow cytometer (FACScan, Becton Dickinson, Heidelberg, Germany). The suspended cells were directly introduced to flow cytometer equipped with an argon laser exciting at 488 nm. For each sample, 5000–10000 events were collected by list-mode data, which consisted of side scatter, forward scatter and fluorescence emissions. CellQuest software (Becton Dickinson) was employed to analyze the list-mode data. Determination of GFP positive events was performed by a standard gating technique. Briefly, the control sample was displayed as a dot plot of GFP signals. The gate was drawn along a line of maximum detected GFP intensity for control samples. The percentage of positive events was calculated as the events within the gate divided by total number of events then subtracting percentage of control samples. In vivo transfection Male BALB/c mice (6–8 weeks old) were provided by the Animal Center, Institute of Health and Epidemic Prevention, Wuhan, China. The mice were housed under normal laboratory conditions (21 ± 2 °C, 12/12-h light–dark cycle) with free access to standard rodent chow and water. Comparison of in vivo transfection efficiency was conducted by subcutaneously injecting liposome/chitosan/DNA complex containing the pVIVO2-GFP plasmid (50 μg total DNA), liposome/DNA complex (50 μg total DNA), naked plasmid DNA (50 μg) into the exposed tibialis anterior muscle bundles mice. 2 days later, the muscle was isolated and fixed in 2% paraformaldahyde. The muscle was cut into several segments with the same length and serial crosssections of 7 cm thickness were cut throughout each segment at −20 °C. One out of each 10 sections was selected for examination under confocal microscope and the representative images were reported. Cytotoxicity assays Cytotoxicity of liposome/chitosan/DNA ternary complexes was evaluated by the 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-Diphenyltetrazolium bromide (MTT) assay (10). Cells were seeded at a cell density of 5 × 103 cells/well in 96-well plates and incubated for 24 h. Liposome/DNA lipoplex or liposome/chitosan/DNA ternary complexes were added to the cells in the presence of 10% FBS for 24 h. At the end of the transfection experiments, cells were washed with PBS and then 20 μl of 5 mg/ml MTT solution was added to the cells in each well. Plates were incubated for an additional 2 h at 37 °C. The medium containing MTT was removed and 150 μl of DMSO was added to dissolve the formazan crystals formed by living cells. Absorbance was measured at 490 nm
FIG. 2. Binding behavior of liposome/chitosan/DNA studied by competitive binding studies using [Ru(phen)2dppz]2+. Error bars indicate SD among three individual experiments. Groups 1–7: the N/P ratio of the ternary complexes was 3:0:1, 3:0.3:1, 3:0.6:1, 3:0.9:1, 3:1.2:1, 3:1.5:1 and 3:2:1, respectively.
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FIG. 3. Effect of chitosan on the particle size (A) and zeta potential (B) of liposome/ chitosan/DNA complexes. Complexes were prepared with 50 μg/ml DNA in 10 mM Tris buffer, pH 8.0. Error bars indicate SD among three individual experiments. Groups 1–5: the N/P ratio of the ternary complexes was 3:0:1, 3:0.3:1, 3:0.9:1, 3:1.5:1 and 3:2:1, respectively. Group 6: the N/P ratio of the liposome/polylysine/DNA complex was 3:2:1. *P b 0.05, **P b 0.01 compared with group 1.
using a Labsystems iEMS microplate reader. Results are expressed as the percentage viability with respect to control cell cultures with no complexes added. Statistics Experiments were carried out with three or four replicates. Statistical analyses were performed by Student's t test. Values with P b 0.05 are considered significant.
RESULTS Electrophoresis of the complexes By using an agarose gel electrophoresis technique, the ability of liposome, chitosan and liposome/chitosan complex to interact with DNA was investigated. Consistent with other group's data, DOTAP: DOPE liposome showed strong ability to couple DNA (Fig. 1A) due to electrostatic interaction between liposome and DNA. The extent of DNA retardation in the
FIG. 5. Transfection efficiency of liposome/chitosan/DNA nanoparticles incubated with HepG2 (A) and NIH3T3 (B) cells in the presence or absence of serum. Liposome/chitosan/ DNA nanoparticles containing 1.5 μg of DNA with different N/P ratios were added to the cells and GFP fluorescence was determined by flow cytometric analysis. Error bars indicate SD among three individual experiments. Groups 1–7: the N/P ratio of the ternary complexes was 3:0:1, 3:0.3:1, 3:0.6:1, 3:0.9:1, 3:1.2:1, 3:1.5:1 and 3:2:1, respectively. Group 8: the N/P ratio of the liposome/poly-L-lysine/DNA complex was 3:2:1.
sample well increased with the increasing ratio of liposome/DNA. The DNA was completely retarded in the well but not migrated into the gel when the liposome/DNA ratio comes to 2:1. As expected, chitosan condenses DNA very well. The migration of DNA is retarded completely when the chitosan/DNA ratio is higher than 1.5 (Fig. 1B). However DNA was completely retarded in the well in the liposome/chitosan/DNA ternary complex and the EtBr fluorescence intensity decreased gradually with the addition of chitosan (Fig. 1C).
FIG. 4. Transmission electron microscopy of liposome/chitosan/DNA complex. Liposome/chitosan/DNA complex was prepared at 3:2:1 charge ratio. B is higher magnification of the corresponding images of A. The internal scales are 200 nm (A) and 20 nm (B) respectively.
VOL. 111, 2011 The reason may be that chitosan condenses DNA and then forms a compact complex with liposome which results in the inability of EtBr to intercalate into the DNA double helix. Competitive binding assay The electrostatic nature of interaction of liposome/chitosan/DNA can be examined by competitive binding studies using [Ru(phen)2dppz]2+ as DNA stain. After combination with DNA, the [Ru(phen)2dppz]2+ binds strongly to DNA by intercalating the large aromatic dipyridophenazine (dppz) ring from the direction of the major groove in between the base pairs and results in a remarkable enhancement in luminescence intensity (11). This combination is reversible and can be replaced by other stronger interaction. As shown in Fig. 2, when the DNA/[Ru(phen)2dppz]2+ complex was mixed with liposome/chitosan, the fluorescence intensity of the solution decreased as a result of competitive binding of liposome/chitosan to DNA. The fluorescence decreased further along with the increase of chitosan. Particle size and zeta potential of liposome/chitosan/DNA complexes Since the size and zeta potential of the complexes are important parameters for in vivo and in vitro transfection efficiency, the size and zeta potential of the liposome/chitosan/DNA ternary
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complexes were determined and compared with those of the corresponding lipoplexes. As shown in Fig. 3, the particle size and zeta-potential of the ternary complexes were dependent on the given chitosan/DNA ratios. Following addition of chitosan, the surface potential of the complexes increased gradually. However, the particle size increased at the initial stage because chitosan was distributed at the interface of DNA and the positive charges were insufficient to condense DNA. With the continuous addition of chitosan, DNA was condensed sufficiently to form compact and stable complexes of about 120 nm. The effect of chitosan on the DNA condensing activity and zeta potential in ternary complex was found similar with that of polyL-lysine, a positive control. Transmission electron microscopy (TEM) In order to assess the size and morphology of the liposome/chitosan/DNA complexes, TEM technique was employed. As shown in Fig. 4, the mean size of the liposome/chitosan/DNA complex at 3:2:1 charge ratio is inferior to 150 nm, which confirms the data characterized by photon correlation spectroscopy. The complex appears spherical with a homogenous distribution of DNA within the particles.
FIG. 6. GFP expression in mouse tibialis anterior muscles. After subcutaneous administration of liposome/chitosan/DNA complexes, the muscle was isolated and cut into 7 segments after 2 days. GFP was observed under confocal microscopy. A showed the representative image. a: naked DNA; b–e: the N/P ratio of the ternary complexes was 3:0:1, 3:0.3:1, 3:0.9:1 and 3:1.5:1, respectively. The internal scale is 300 μm. B: GFP expression was quantified.
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In vitro transfection of liposome/chitosan/DNA complexes Liposome/chitosan/DNA ternary complexes were assessed for their in vitro transfection efficiency by monitoring the transient expression of GFP in HepG2 and NIH3T3 cells. HepG2 and NIH3T3 cells were transfected using ternary complexes with different ratios of chitosan/ DNA and GFP expression was determined by flow cytometry. Consistent with the previous data, poly-L-lysine significantly enhanced the transfection efficiency of liposome in the presence or absence of serum. The overall transfection efficacy of liposome/ chitosan/DNA ternary complexes was also dramatically higher than that of lipoplex. The expression levels are dependent on the ratio of chitosan/DNA in ternary complexes. When the N/P ratio comes to 3:2:1, no significant difference in transfection efficiency between liposome/chitosan/DNA and liposome/poly-L-lysine ternary complex was found (Fig. 5). In vivo transfection To determine whether the vitro results could be compatible with in vivo experiments, we assessed the potential of liposome/chitosan/DNA nanoparticles to transfer genes into the tibial anterior muscle. Two days after injection of liposome/ chitosan/DNA nanoparticles, the expression of GFP was monitored using confocal microscopy. As shown in Fig. 6, naked DNA without carriers shows very low transfection efficiency. The transfection efficiency of liposome/DNA complex exceeded that of naked DNA. Upon increasing chitosan in liposome/chitosan/DNA nanoparticles, the expression of GFP significantly increased. Cell viability For eventual in vivo application, a required characteristic of gene delivery systems is that of low cytotoxicity. The application of cationic liposome and treatment with plasmid DNA has often suffered from serious cytotoxic side effects. To determine whether chitosan affects the toxicity of liposome, the effect of the liposome/chitosan/DNA complexes on cell viability was determined using the MTT assay (Fig. 7). Consistent with previous data (12), there is a decrease in cell viability when HepG2 cells and NIH3T3 cells are incubated with lipoplex. Poly-L-lysine further increases the cell toxicity of liposome. However when the cells were treated with liposome/chitosan/DNA ternary complexes, no cytotoxicity was found in these cells, suggesting that chitosan condensation could reduce the cytotoxicity of liposome. DISCUSSION A number of non-viral vectors have been developed for gene therapy because of their higher safety and lower cost. Liposomes and cationic polymers are two major classes of non-viral gene delivery carriers. Although liposomes formed from cationic phospholipids offer several advantages over viral gene transfer, e.g. low immunogenicity and ease of preparation (13), the success of the liposomal approach is limited due to their serious cytotoxic side effects, low transfection efficiency and instability in the serum. Current efforts to improve the transfection efficiency of cationic liposomes focus on the synthesis of new cationic lipids and on the search for better cationic liposome formulations (14). The addition of a polycation, such as polyethylenimine (PEI), protamine sulfate, poly-L-lysine or spermine, has been shown to enhance liposome-mediated gene transfection. This is thought to occur due to electrostatic interactions between the polycation and DNA, resulting in a charge neutralization of the complex and the formation of a condensed structure. It has been shown that some liposome/DNA complexes showed “spaghetti and meatballs” structures (15), suggesting that the DNA molecules are not well condensed in the complexes and they may exist in an extended conformation covered by lipids. In contrast to liposome/DNA complex, the condensed structure of the liposome/polycation/DNA complexes, due to its diminished size, may be more readily endocytosed by the cells and result in the increased levels of transgene expression. However the polycationic nature of these
FIG. 7. Cytotoxicity of liposome/chitosan/DNA nanoparticles on the HepG2 (A) and NIH3T3 (B) cells. Liposome/chitosan/DNA nanoparticles containing 0.5 μg of DNA with different N/P ratios were added to the cells and the toxicity was determined using the MTT assay. Error bars indicate SD among three individual experiments. Group 1: Control group. Groups 2–8: The N/P ratio of the ternary complexes was 3:0:1, 3:0.3:1, 3:0.6:1, 3:0.9:1, 3:1.2:1, 3:1.5:1 and 3:2:1, respectively. Group 9: The N/P ratio of the liposome/poly-L-lysine/DNA complex was 3:2:1. *P b 0.05 compared with group 1.
polycations (e.g. PEI and poly-L-lysine) appeared to be the main origin of their marked toxicity. The toxicity has severely limited their use as gene delivery vectors in vivo. Chitosan is a cheap, biocompatible, biodegradable and non-toxic cationic polymer that forms polyelectrolyte complexes with DNA and considered to be a good candidate for a gene delivery carrier (7). Although chitosan successfully transfected cells in vitro, the transfection efficiency showed to be much lower than that of other cationic polymer vehicles such as PEI (5). Therefore, the combination of liposome and chitosan might be a promising approach for enhancing transfection efficiency. In this study the ability of chitosan as a DNA condensing agent was already shown in Fig. 3 when the size of transfection complex had decreased due to chitosan-added condensation of plasmid DNA. Significant differences were observed in the transfection efficiency of liposome/chitosan/DNA complexes and the charge ratio of chitosan to DNA affected the transfection efficiency of the complexes. The in vitro and in vivo transfection activities of the resulting liposome/chitosan/ DNA ternary complexes are significantly higher than that of the liposome/DNA complex. One possible reason for different transfection efficiency is the difference in their DNA condensing ability. However we could not rule out the possibility that the increased uptake of the complexes also contributed to the enhanced transfection activity
VOL. 111, 2011 considering the increased positive charge of the complexes with increasing chitosan content of the complexes. In this study poly-Llysine was used as a positive control to compare the effect of chitosan on the transfection efficiency of liposome. We found that chitosan showed a similar DNA condensing activity and induced a high transfection efficiency of liposome to a similar extent with poly-L-lysine. It has been reported that chitosan showed significantly lower toxicity than poly-L-lysine and PEI (16,17). In this study the toxicity of the liposome/DNA binary complex and liposome/chitosan/DNA ternary complexes on NIH3T3 and HepG2 cells was evaluated using MTT assay. Consistent with the previous data, liposome/DNA binary complex showed significant cell toxicity and NIH3T3 cells are more sensitive to the toxicity of the liposome/DNA complex than HepG2 cells. Liposome/ poly-L-lysine/DNA ternary complex was more toxic to the cells than liposome/DNA binary complex. In contrast, the liposome/chitosan/DNA ternary complex did not exhibit any noticeable cell toxicity. This probably suggests that chitosan is either acting as a detoxifying agent to reduce the toxicity of liposome/DNA complex or the reduced complex size may have been attributed to lesser cytotoxic effect (18). In the presence of an appropriate amount of chitosan, successful transfection can be achieved with suboptimal ratios of cationic liposomes. In conclusion, we present the development and characterization of a new strategy to prepare more efficient and safe liposome/chitosan/ DNA complexes as an effort to further improve the performance of cationic liposome. These small-sized liposome/chitosan/DNA ternary complexes represent a new class of nonviral gene delivery vehicles that might be useful in gene therapy. However, further understanding of the detailed mechanism underlying the synergism is necessary to improve the design of future gene transfer carriers. References 1. El-Aneed, A.: An overview of current delivery systems in cancer gene therapy, J. Control. Release, 94, 1–14 (2004). 2. Morille, M., Passirani, C., Vonarbourg, A., Clavreul, A., and Benoit, J.: Progress in developing cationic vectors for non-viral systemic gene therapy against cancer, Biomaterials, 29, 3477–3496 (2008).
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3. Gao, X. and Huang, L.: Potentiation of cationic liposome-mediated gene delivery by polycations, Biochemistry, 35, 1027–1036 (1996). 4. Li, S., Rizzo, M., Bhattacharya, S., and Huang, L.: Characterization of cationic lipid– protamine–DNA (LPD) complexes for intravenous gene delivery, Gene Ther., 5, 930–937 (1998). 5. MacLaughlin, F., Mumper, R., Wang, J., Tagliaferri, J., Gill, L., Hinchcliffe, M., and Rolland, A.: Chitosan and depolymerized chitosan oligomers as condensing carriers for in vivo plasmid delivery, J. Control. Release, 56, 259–272 (1998). 6. Rege, P., Shukla, D., and Block, L.: Chitinosans as tableting excipients for modified release delivery systems, Int. J. Pharm., 181, 49–60 (1999). 7. Lai, W. and Lin, M.: Nucleic acid delivery with chitosan and its derivatives, J. Control. Release, 134, 158–168 (2009). 8. Zhao, Q., Chen, J., Han, M., Liang, W., Tabata, Y., and Gao, J.: Combination of poly (ethylenimine) and chitosan induces high gene transfection and low cytotoxicity, J. Biosci. Bioeng., 105, 65–68 (2008). 9. Mirska, D., Schirmer, K., Funari, S., Langner, A., Dobner, B., and Brezesinski, G.: Biophysical and biochemical properties of a binary lipid mixture for DNA transfection, Colloids Surf. B Biointerfaces, 40, 51–59 (2005). 10. Hanelt, M., Gareis, M., and Kollarczik, B.: Cytotoxicity of mycotoxins evaluated by the MTT-i assay, Mycopathologia, 128, 167–174 (1994). 11. Biver, T., Secco, F., and Venturini, M.: Mechanistic aspects of the interaction of intercalating metal complexes with nucleic acids, Coord. Chem. Rev., 252, 1163–1177 (2008). 12. Resina, S., Prevot, P., and Thierry, A.: Physico-chemical characteristics of lipoplexes influence cell uptake mechanisms and transfection efficacy, PLoS ONE, 4, e6058 (2009). 13. Uddin, S.: Cationic lipids used in non-viral gene delivery systems, Biotech. Mol. Biol. Rev., 2, 058–067 (2007). 14. Kim, T., Chung, H., Kwon, I., Sung, H., Shin, B., and Jeong, S.: Polycations enhance emulsion-mediated in vitro and in vivo transfection, Int. J. Pharm., 295, 35–45 (2005). 15. Sternberg, B., Sorgi, F., and Huang, L.: New structures in complex formation between DNA and cationic liposomes visualized by freeze-fracture electron microscopy, FEBS Lett., 356, 361–366 (1994). 16. Erbacher, P., Zou, S., and Remy, J.: Chitosan-based vector/DNA complexes for gene delivery: biophysical characteristics and transfection ability, Pharm. Res., 15, 1332–1339 (1998). 17. Roy, M., Mao, H., and Leong, K.: DNA-chitosan nanospheres: transfection efficacy and cellular uptake, Proc. Int'l. Symp. Control. Rel. Bioact. Mater., 24, 673–674 (1997). 18. Lee, M., Chun, S., Choi, W., Kim, J., Choi, S., Kim, A., Oungbho, K., Park, J., Ahn, W., and Kim, C.: The use of chitosan as a condensing agent to enhance emulsion mediated gene transfer, Biomaterials, 26, 2147–2156 (2005).