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tripolyphosphate nanoparticles and application as a gene carrier for targeting SMMC7721 cells
Journal of Bioscience and Bioengineering VOL. 111 No. 6, 719 – 724, 2011 www.elsevier.com/locate/jbiosc
Preparation of galactosylated chitosan/tripolyphosphate nanoparticles and application as a gene carrier for targeting SMMC7721 cells Hui Jiang, Hong Wu, Ying-long Xu, Jing-zhou Wang, and Yong Zeng⁎ Department of Hepato-bilio-pancreatology Surgery, West China Hospital, Sichuan University, Chengdu 610041, Sichuan Province, China Received 25 November 2010; accepted 17 January 2011 Available online 22 February 2011
Nanoparticles composed of galactosylated chitosan (GC) and tripolyphosphate (TPP) were prepared and their application as potential gene carriers for targeting SMMC7721 cells was investigated. The results showed that at certain pH (5.5–6.2) of GC solutions, small and stable nanoparticles were obtained at an optimal weight ratio of 5:1 (GC/TPP). Transmission electron microscope (TEM) revealed formation of spherical particles. The optimal pH of cell culture environment for transfection was from 6.4 to 6.7, which was the same pH as the polymer complex formation of GC/TPP solutions. The charge ratio of GC/TPP to DNA (N/P) at 10:1, 20:1 and 30:1 were checked for transfection and under optimized conditions, the GC/TPP-DNA nanoparticles successfully transfected 6.8% of the SMMC7721 cells as represented by overexpression of enhanced green fluorescent protein (EGFP), which showed a much more higher efficiency when compared to 0.6% of GC/DNA transfection under the same conditions. The presented results indicate that the GC/TPP nanoparticles might be very attractive to be used as a gene delivery carrier for hepatocyte targeting, thus warranting further in vivo or clinical investigations. © 2011, The Society for Biotechnology, Japan. All rights reserved. [Key words: Galactosylated chitosan; Tripolyphosphate; Gene delivery; Transfection efficiency]
Gene therapy, a technique used for correcting faulty genes involved in diseases, is a promising approach for inherited or acquired diseases, like severe combined immune deficiency and cancer (1). The key issue of this technology is to deliver the therapeutic gene into its target sites and to make it function. Extensive efforts have been made for this purpose and different delivery systems, including virus-mediated and non-viral gene transfer techniques, have been developed aiming at an efficient and safe gene delivery (2–4). Despite great progress in the past decades in using viral vectors which represent a highly efficient and commonly used method, their application is always concerned with their biological safety (5). Therefore, non-viral alternatives, like liposomes, cationic polyplexes, microparticles and nanoparticles, have been developed as safer strategies for gene delivery, although with lower efficiency when compared with viral-system (6). The low effectiveness of non-viral gene delivery systems is mainly due to the existence of extracellular and intracellular barriers that they have to pass through. These barriers also make the delivered genes to get more susceptible to degradation, which must be avoided before it reaches the target cells. Instead, the vectors must be degraded after reaching their destination. At these points, chitosan (a cationic polymer) and chitosan derivatives attract great interests as non-viral vectors due to their biocompatibility, low cytotoxicity, low immunogenicity, ability to protect DNA against DNase degradation and biodegradability (7–9). Extensive efforts have been made to improve the delivery efficiency and cell specificity of chitosan-based method, such as introduction of
⁎ Corresponding author. Tel./fax: +86 28 85422475. E-mail address: [email protected] (Y. Zeng).
galactose, transferrin, folic acid and mannose (7). Among these improvements, introduction of galactose into chitosan to produce galactosylated chitosan (GC) as a carrier for gene delivery has been successfully used to transfect a wide range of cell lines (10–12), which, for example, takes advantage of the interaction between the galactose residues conjugated on chitosan molecule and the asialoglycoprotein receptors abundantly expressed on the surface of hepatoma cells aimed at the treatment of liver cancers (13–15). Importantly, previous reports have demonstrated that treatment of cells with GC promotes the cell viability or negligible toxicity (10,11). However, the transfection efficiency of GC-based system is still not so satisfactory due to several lines of limitations (16), for example, DNA association to chitosan. It was shown that tripolyphosphate (TPP) is very helpful in encapsulating and sustaining the release of active molecules (16,17) including DNA as reported by Csaba et al. coupled with chitosan and tested with human embryonic kidney 293 cells (16). Because tripolyphosphate (TPP) is a crosslinking agent containing anions, it interacts with the amino-groups (cation) of chitosan and improves the intra- and inter molecular interactions of GC and TPP, which contributes to stable and homogenous nanoparticle formation (16). Therefore, in the present study we tested the conjugation of TPP with GC (50 kDa) to produce GC/TPP nanoparticles aiming at a relatively higher transfection efficiency. Preparation of GC/TPP nanoparticles was developed and optimized. Their size and zeta potential were determined by dynamic light scattering (DLS) and zetasizer measurements, respectively. The morphology of the nanoparticles was imaged by transmission electron microscopy (TEM). The nanoparticles were tested to transfect SMMC7721 cells and the efficiency was measured by fluorescent microscopy and fluorescence-activated cell sorting analysis (FACS).
1389-1723/$ - see front matter © 2011, The Society for Biotechnology, Japan. All rights reserved. doi:10.1016/j.jbiosc.2011.01.012
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Materials Galactosylated chitosan (molecular weight, 50 kDa; deacetylation degree, 90%; galactosylation degree, 9.8%) was kindly provided by Prof. Y. Li (College of Chemistry, Sichuan University, China). Detailed synthesis and characteristics of GC has been illustrated (10,11). Pentasodium tripolyphosphate(TPP) was purchased from Sigma. The plasmid pEGFP-N1 (4.7 kb) encoding enhanced green fluorescence protein was a gift from Dr. Dan Long and the cell line SMMC7721 was obtained from Laboratory of Transplant Engineering and Immunology, West China Hospital. DNA extraction pEGFP-N1 plasmid was amplified in Escherichia coli DH5α and was purified by use of Plasmid Maxi Kit (Tiangen Biotech Co., Ltd). The purified plasmid DNA was dissolved in PBS at a concentration of 10 μg/ml and stands for 12 h at room temperature before use. The pH of PBS was adjusted according to the GC/TPP solution as indicated (see below). Nanoparticle preparation To prepare GC/TPP nanoparticles, GC was dissolved in 150 mM NaCl at a final concentration of 1 mg/ml with magnetic stirring and the pH was adjusted to required value with 1 M NaOH and 1 M HCl. TPP was dissolved in H2O at a concentration of 0.5 mg/ml then adjusted to pH 8.9. After sterilization by filtration through a 0.22 μm filter, GC/TPP nanoparticles were prepared by use of ion-crosslinking method according to Bodmeier et al. (18) and the pH were 4.8, 5.0, 5.2, 5.5, 5.8, 6.0, and 6.2, respectively. The weight ratio of GC to TPP was 5:1. GC/TPP mixtures were heated in a water bath at 65–70°C for 10 min then vortexed. The GC/TPP mixture stands at room temperature for at least 30 min before incorporation of DNA. To prepare DNA-GC/TPP nanoparticles (DNA loading), DNA was loaded into GC/TPP nanoparticles through electrostatic interactions between DNA and GC/TPP nanoparticles by incubating DNA with GC/TPP polymer complex solutions for 20 min. The ratios of GC to plasmid DNA (N/P = w/w) were 3:1, 5:1, 10:1, 20:1, and 30:1 respectively. Finally, appropriate PBS was added into the mixture prior to the transfection assays. The pH of PBS was adjusted respectively according to the pH value of GC/TPP polymer complex solutions. Measurements of Size, morphology and zeta potential of nanoparticles The morphology of the nanoparticles was examined using a transmission electron microscope (TEM: H-7651, Japan) and measured by granule diameter. Appropriate amounts of sample were placed on a copper grill covered with nitrocellulose then dried at room temperature. After negatively staining with tungsten phosphate, they were examined with electron microscopy. The particle size and zeta potential of GC/TPP nanoparticle were measured by dynamic light scattering (DLS) particle size analyzer
J. BIOSCI. BIOENG., (LB550, Horiba Instruments Inc., Horiba, Japan) and a 3000HS/IHPL Zetasizer (Malvern Instruments Ltd., Malvern, UK), respectively. Agarose gel electrophoresis The DNA binding ability of GC/TPP nanoparticles was evaluated by agarose gel electrophoresis. The nanoparticle solutions of pEGFP with GC/TPP polymer were loaded into individual wells of 1% agarose gel and separated at 100 V for 40 min, then stained with 0.5 g/ml ethidium bromide. The resulting plasmid migration pattern was revealed under UV light. Cell Culture and transfection The hepatoma cell line SMMC7721 cells were cultured in RPMI1640 standard growth medium containing 10% fetal bovine serum (FBS) at 37°C under 5% CO2 atmosphere. After the cells reached 50% of confluence, the culture was diluted 1:8 for transfection. Cells were plated at 5 × 104 cells/well containing 500 μl complete medium in clear 24 well tissue plates. After incubation for 24 h, the medium was discarded and the cells were washed once with PBS at the same pH used for transfection as well as with the GC/TPP solution. Nanoparticles containing DNA solution were added to the cells at a concentration of 0.3 μg/well and incubated for 4–5 h. Finally, the solution was discarded and fresh complete medium was added to the cells again. Fluorescene microscopy Fluorescence microscope images were recorded with a DP controller software at 24 and 48 h after transfection. An inverted microscope (IX71, Olympus; Japan) equipped with a high pressure mercury lamp (CST-5000C, Japan) and an appropriate set of filters for fluorescein (FITC) excitation and emission wavelengths were used. Flourescence-activated cell sorting analysis (FACS) After transfection and incubation at 37°C for 48 h, the hepatoma cells were first checked for GFP overexpression by use of fluorescent microscope described above, then the cells were washed with PBS first and followed by a treatment of 0.25% trypsin and 0.02% EDTA (Beyotime, China) until detachment of the cells. Then 1 ml of complete medium was added and the mixture was centrifuged. Finally the cells were washed again with PBS at physiological pH and replenished. Finally, the samples were transferred to FACS tubes and analyzed directly. For each sample, 1 × 106 cells diluted with 300 μl PBS were counted and analyzed with BD FACSDiva software using appropriate controls and gates. Dead cells and cell debris were excluded from the calculation for the percentage of cells expressing GFP. Statistical analysis Each experiment was performed in triplicate and the value refers to the mean ± standard deviation. A SPSS software package (v.16) was used for statistical analyses. For assessment of the statistically significant homogeneous subset, one-way ANOVA tests were performed followed by a Student–Newman–Keuls ranking with a confidence level of α ≤ 0.05.
FIG. 1. The size and zeta potential of GC/TPP nanoparticles at different pH of GC solutions. (A) Effects of the pH of GC solutions on the size of GC/TPP nanoparticles (w/w = 5:1). (B) Comparison of the diameter of GC/TPP and GC/TPP-DNA nanoparticles (N/P = 20:1). (C) Zeta potential of GC/TPP complex. (D) pH of the GC/TPP complex solution.
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TABLE 1. The average diameter and zeta potential of GC-TPP/DNA under various N/P ratios (the pH of GC solution is 6.0 at 25°C). N/P ratio (w/w) 10:1 20:1 30:1
Z-average (nm)
Zeta potential (mV)
279 226 185
11.8 14.4 22.6
RESULTS Characterization of GC/TPP nanoparticles Several factors, such as pH, molecular weight of chitosan and its deacetylation level, all affect the physical and chemical properties of nanoparticles, which further influence their stability, loading ability of DNA and the transfection efficiency of the nanoparticles (19–22). Therefore, we first characterized the physical properties of GC/TPP nanoparticles. As shown in Fig. 1A, an increase of the pH value of the GC solution for particle preparation results in the formation of significantly smaller GC/TPP nanoparticles, which is believed to increase the solubility of particles at physiological pH and improve the efficiency of DNA release (23–25). However when pH exceeded 6.2, GC solution was turbid and could not form nanoparticles with TPP. Loading of pEGFP DNA into the nanoparticles slightly increased the size of particles in contrast to the empty GC/TPP nanoparticles (Fig. 1B). To get an idea about the stability of GC/TPP nanoparticles, the zeta potential, which indicates the present repulsive force and is widely used to predict the long-term stability of the particles, was determined. As shown in Fig. 1C, under all the conditions the nanoparticles show high values of positive zeta potential, which suggest that they tend to repel each other and not come together therefore representing a nice longterm stability under tested conditions. Furthermore, no drastic differences of the zeta potential with various pH ranging from 4.8 to 6.2 were observed. In addition, the pH of polymer complex, as shown in Fig. 1D, ranged from 5.8 to 7.1 according to pH of GC (4.8–6.2) and TPP (8.9) thus further indicates the stable property of GC/TPP nanoparticles. Another parameter, the N/P ratio (i.e., the ratio between cationic polymers and DNA, w/w), is believed to play an important role in affecting the degree of complexation like particle diameter, thus further influencing the transfection efficiency and cytotoxicity of carriers. A low N/P ratio would yield physically unstable particle/DNA complexes which leads to poor transfection efficiency due to low amounts of DNA delivery, while those too stable complexes also result in the same problem if N/P ratio is too high because DNA cannot be released (9,26). As shown in Table 1, with increased N/P ratio, the mean size of the nanoparticles decreased and reaches a minimum value of 185 nm at the N/P ratio of 30:1. Correspondingly, the zeta potential increased with the increase of the N/P ratio, suggesting stable particle/DNA complexes formed. The complex formation of GC/TPP nanoparticle with DNA was further evaluated by gel retardation assay, in which nanoparticle/DNA solutions were electrophoresed on agarose gels and visualized with ethidium bromide (Fig. 2A). Condensed DNA by GC/TPP particles remained at the top of the agarose gel at N/P ratio of 10, 20 and 30 (Fig. 2A, lanes 4–6), suggesting DNA was successfully entrapped into GC/TPP nanoparticles. In contrast, unconjugated DNA (Fig. 2A, lane 1) runs into the gel as well as those with low N/P ratios (Fig. 2A, lanes 2 and 3) due to their unstable states migrate to the positive pole. Fig. 2B shows a TEM photograph of representative GC/TPP-DNA nanoparticles and a spherical and polydisperse nature of the particles is obvious. The particle size distribution was achieved though DLS (LB-550) and was shown in Fig. 3, which indicates that loading of DNA slightly increases the particle size (compare panels A and B in Fig. 3). Transfection studies To test the effectiveness of the prepared GC/TPP-DNA nanoparticles, transfection studies were performed by use of the hepatoma cell line SMMC7721. As shown in Fig. 4, the
FIG. 2. (A) Agarose gel retardation analysis of GC/TPP-DNA nanoparticles. GC/TPP nanoparticles were combined with DNA at different N/P ratios. Lane 1: naked DNA; lane 2: N/P = 3; lane 3: N/P = 5; lane 4: N/P = 10; lane 5: N/P = 20; lane 6: N/P = 30. The gel was 1% and stained with ethidium bromide. (B) TEM photograph of GC/TPP nanoparticles (200 nm).
transfection efficiency varied significantly at different pH values of the cell culture and the highest transfection efficiency was achieved at pH of 6.7 (Fig. 4A). The transfection efficiency varied also at different N/P ratio as analyzed with FACS, and in which, N/P ratio of 20:1 gave the best efficiency compared with 10:1 and 30:1 (Fig. 4B). This result was further confirmed by use of fluorescent microscopy imaging (Fig. 5). A decrease in the transfection efficiency was further observed at other pH values and N/P (data not shown). Most importantly, no transfection was observed when naked DNA was used (Fig. 5A) which further suggests the transfection specificity of the GC/TPP-DNA nanoparticles observed. Furthermore, the pEGFP expression from FACS analysis clearly demonstrated a higher transfection efficiency of the SMMC7721 cell population with GC/ TPP-DNA nanoparticles at the optimal N/P ratio (i.e., 20:1) (with an efficiency of 6.8 ± 0.9%) when compared with the GC/DNA complexes at the same N/P ratio (the efficiency is 0.6 ± 0.2%). Even at nonoptimal N/P ratio of 10:1 and 30:1, the transfection efficiency still reaches 4.6 ± 0.7% and 5.9 ± 0.4%, respectively, which is 7–10 times higher than GC-DNA particles, further illustrating the effective “helper” effect of TPP addition.
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FIG. 4. Optimization of in vitro transfection parameters (0.5 μg of DNA per well and an incubation time of 4 h). (A) The pH effects (at N/P ratio of 20:1). (B) Influence of N/P ratios (the pH of transfection medium was 6.7) and comparison of GC/TPP-DNA, GC-DNA and naked DNA.
FIG. 3. The diameter distributions of nanoparticles observed by DLS (LB550) with pH of GC solution at 6.0. (A) GC/TPP: the mean diameter was 163.5 nm. (B) GC/TPP-DNA: the mean diameter was 198.6 nm, with an N/P ratio of 30:1.
DISCUSSION Due to the abundant expression of asialoglycoprotein (ASGP) in hepatoma cells, drugs or gene carriers conjugated with a ligand has been used to target ASGP receptors for the treatment of liver cancers (13,14). GC is a kind of ASGR based gene carrier to target liver cells. Although several reports already indicate that GC, including other cationic polymers, has the potential to target hepatocytes (27,28), there are few reports thoroughly investigating the transfection efficiency of galactosylated nanoparticles. Furthermore, TPP improves the transfection efficacy of chitosan (16) but has not been tested by use of GC. Therefore, the aim of the present work was to evaluate the conjugation of TPP into GC to test its efficacy as a gene carrier for HepG2 cells targeting. Our results now demonstrate that factors determining a high transfection efficiency depend on the pH of the cell culture, the charge ratio of GC to DNA as well as the preparation means of the nanoparticles. We clearly demonstrate that the size and the zeta potential of GC/ TPP nanoparticles varied in accordance with the pH value of the GC solutions used and an increase of the pH value results in the formation of significantly smaller GC/TPP nanoparticles (Fig. 1). This observation could be explained by the decrease of positive charges of GC while the pH increases, which leads to a reduction of the charge interactions among nanoparticles. After DNA loading, the nanoparticle size increased
slightly. Moreover by complexing GC and TPP, small and stable nanoparticles were achieved leading to an improvement of the transfection efficiency. Furthermore, the characteristics of the nanocarrier system are not the sole determinants of the transfection efficiency since the properties, particularly the pH value, of the cell culture also play an important role (Fig. 4). A higher transfection efficiency was achieved between the pH values of 6.4 and 6.7 and a drastic decrease was observed towards higher pH while a relatively gradual decrease towards the acidic pH, which is correlating with previous reports, i.e. a slightly acidic culture medium is beneficial for a higher transfection efficiency (25,29). This phenomenon is due to the protonation of amine groups of GC under slight acidic conditions which facilitates the binding of negatively charged DNA as well as the DNA condensation into particles through phase separation. However, too acidic pH is not helpful to improve the transfection efficiency because of the strong electrostatic interactions between the negatively charged DNA and the positively charged nanoparticles, which ultimately inhibits DNA release (30). On the other hand, dramatic decrease of the transfection efficiency at higher pH might be a consequence of DNA dissociation from the nanoparticles (31). In addition, the pH of the cell culture should be the same as the GC/TPP polymer complex solutions, otherwise the size of nanoparticles would be changed. Therefore, the composition of the medium should be carefully selected in order to avoid aggregation which ultimately affects the transfection efficiency and the distribution within the cell (22). For example, a simple medium (RPMI1640) or a complete medium (RPMI1640 containing 10% FBS) leads to aggregation of GC/TPP-DNA nanoparticles, forming large particles with diameters ranging from 1 to 2 μm, irrespective of ultrasound dispersion or magnetic stirring
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Furthermore, in vivo or clinical experiments should also be performed to test the system as a novel hepatocyte-targeting gene carrier for its safety, novelty as well as applicability for medical purposes and this work is ongoing in our lab. ACKNOWLEDGMENTS This work was supported by the Foundation of Key project of Sichuan Province (No. 07SG004004). We gratefully acknowledge Prof. Ying Li for kindly providing GC, Prof. Shengfu Li for valuable assistance in microscopic analysis and Dr. Yangyan He for helps in FACS analysis. REFERENCES
FIG. 5. Fluorescent microscopy imaging of transfected cells under optimized parameters. Fluorescent microscopy images were randomly taken from a subset of transfected cells with (A) N:P = 10:1, (B) N:P = 30:1, (C) N:P = 20:1. Transfection experiments were performed in PBS at pH of 6.7, with 0.3 μg per well of pEGFP and incubated for 4 h. Image was taken after 48 h of transfection.
treatment. Of course, no transfection was obtained since there is no cellular uptake of these huge particles. Finally, the N/P ratio is another important factor that affects transfection efficiency (Fig. 4) because a low N/P ratio would yield physically unstable complexes and poor transfection, whereas a high N/P ratio results in a poor transfection because of their stability; thus, the DNA cannot be released from the complexes (1). In conclusion, our results demonstrate that GC, as a derivative of chitosan, has the ability to form stable and small nanoparticles when coupled with TPP at optimal pH solutions due to ionic gelation property of TPP (16). And most importantly, the in vitro analysis using SMMC7721 cells suggests that the GC/TPP nanoparticles have a more effective transfection efficiency when compared with GC itself, thus warranting further improvement of the system. At this point, factors like the degree of deacetylation and molecule weight (MW) of chitosan, pH, serum, charge ratio of chitosan to DNA and cell type and so on all have to be optimized to enhance the transfection efficiency.
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Preparation of galactosylated chitosan/tripolyphosphate nanoparticles and application as a gene carrier for targeting SMMC7721 cells Hui Jiang, Hong Wu, Ying-long Xu, Jing-zhou Wang, and Yong Zeng⁎ Department of Hepato-bilio-pancreatology Surgery, West China Hospital, Sichuan University, Chengdu 610041, Sichuan Province, China Received 25 November 2010; accepted 17 January 2011 Available online 22 February 2011
Nanoparticles composed of galactosylated chitosan (GC) and tripolyphosphate (TPP) were prepared and their application as potential gene carriers for targeting SMMC7721 cells was investigated. The results showed that at certain pH (5.5–6.2) of GC solutions, small and stable nanoparticles were obtained at an optimal weight ratio of 5:1 (GC/TPP). Transmission electron microscope (TEM) revealed formation of spherical particles. The optimal pH of cell culture environment for transfection was from 6.4 to 6.7, which was the same pH as the polymer complex formation of GC/TPP solutions. The charge ratio of GC/TPP to DNA (N/P) at 10:1, 20:1 and 30:1 were checked for transfection and under optimized conditions, the GC/TPP-DNA nanoparticles successfully transfected 6.8% of the SMMC7721 cells as represented by overexpression of enhanced green fluorescent protein (EGFP), which showed a much more higher efficiency when compared to 0.6% of GC/DNA transfection under the same conditions. The presented results indicate that the GC/TPP nanoparticles might be very attractive to be used as a gene delivery carrier for hepatocyte targeting, thus warranting further in vivo or clinical investigations. © 2011, The Society for Biotechnology, Japan. All rights reserved. [Key words: Galactosylated chitosan; Tripolyphosphate; Gene delivery; Transfection efficiency]
Gene therapy, a technique used for correcting faulty genes involved in diseases, is a promising approach for inherited or acquired diseases, like severe combined immune deficiency and cancer (1). The key issue of this technology is to deliver the therapeutic gene into its target sites and to make it function. Extensive efforts have been made for this purpose and different delivery systems, including virus-mediated and non-viral gene transfer techniques, have been developed aiming at an efficient and safe gene delivery (2–4). Despite great progress in the past decades in using viral vectors which represent a highly efficient and commonly used method, their application is always concerned with their biological safety (5). Therefore, non-viral alternatives, like liposomes, cationic polyplexes, microparticles and nanoparticles, have been developed as safer strategies for gene delivery, although with lower efficiency when compared with viral-system (6). The low effectiveness of non-viral gene delivery systems is mainly due to the existence of extracellular and intracellular barriers that they have to pass through. These barriers also make the delivered genes to get more susceptible to degradation, which must be avoided before it reaches the target cells. Instead, the vectors must be degraded after reaching their destination. At these points, chitosan (a cationic polymer) and chitosan derivatives attract great interests as non-viral vectors due to their biocompatibility, low cytotoxicity, low immunogenicity, ability to protect DNA against DNase degradation and biodegradability (7–9). Extensive efforts have been made to improve the delivery efficiency and cell specificity of chitosan-based method, such as introduction of
⁎ Corresponding author. Tel./fax: +86 28 85422475. E-mail address: [email protected] (Y. Zeng).
galactose, transferrin, folic acid and mannose (7). Among these improvements, introduction of galactose into chitosan to produce galactosylated chitosan (GC) as a carrier for gene delivery has been successfully used to transfect a wide range of cell lines (10–12), which, for example, takes advantage of the interaction between the galactose residues conjugated on chitosan molecule and the asialoglycoprotein receptors abundantly expressed on the surface of hepatoma cells aimed at the treatment of liver cancers (13–15). Importantly, previous reports have demonstrated that treatment of cells with GC promotes the cell viability or negligible toxicity (10,11). However, the transfection efficiency of GC-based system is still not so satisfactory due to several lines of limitations (16), for example, DNA association to chitosan. It was shown that tripolyphosphate (TPP) is very helpful in encapsulating and sustaining the release of active molecules (16,17) including DNA as reported by Csaba et al. coupled with chitosan and tested with human embryonic kidney 293 cells (16). Because tripolyphosphate (TPP) is a crosslinking agent containing anions, it interacts with the amino-groups (cation) of chitosan and improves the intra- and inter molecular interactions of GC and TPP, which contributes to stable and homogenous nanoparticle formation (16). Therefore, in the present study we tested the conjugation of TPP with GC (50 kDa) to produce GC/TPP nanoparticles aiming at a relatively higher transfection efficiency. Preparation of GC/TPP nanoparticles was developed and optimized. Their size and zeta potential were determined by dynamic light scattering (DLS) and zetasizer measurements, respectively. The morphology of the nanoparticles was imaged by transmission electron microscopy (TEM). The nanoparticles were tested to transfect SMMC7721 cells and the efficiency was measured by fluorescent microscopy and fluorescence-activated cell sorting analysis (FACS).
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Materials Galactosylated chitosan (molecular weight, 50 kDa; deacetylation degree, 90%; galactosylation degree, 9.8%) was kindly provided by Prof. Y. Li (College of Chemistry, Sichuan University, China). Detailed synthesis and characteristics of GC has been illustrated (10,11). Pentasodium tripolyphosphate(TPP) was purchased from Sigma. The plasmid pEGFP-N1 (4.7 kb) encoding enhanced green fluorescence protein was a gift from Dr. Dan Long and the cell line SMMC7721 was obtained from Laboratory of Transplant Engineering and Immunology, West China Hospital. DNA extraction pEGFP-N1 plasmid was amplified in Escherichia coli DH5α and was purified by use of Plasmid Maxi Kit (Tiangen Biotech Co., Ltd). The purified plasmid DNA was dissolved in PBS at a concentration of 10 μg/ml and stands for 12 h at room temperature before use. The pH of PBS was adjusted according to the GC/TPP solution as indicated (see below). Nanoparticle preparation To prepare GC/TPP nanoparticles, GC was dissolved in 150 mM NaCl at a final concentration of 1 mg/ml with magnetic stirring and the pH was adjusted to required value with 1 M NaOH and 1 M HCl. TPP was dissolved in H2O at a concentration of 0.5 mg/ml then adjusted to pH 8.9. After sterilization by filtration through a 0.22 μm filter, GC/TPP nanoparticles were prepared by use of ion-crosslinking method according to Bodmeier et al. (18) and the pH were 4.8, 5.0, 5.2, 5.5, 5.8, 6.0, and 6.2, respectively. The weight ratio of GC to TPP was 5:1. GC/TPP mixtures were heated in a water bath at 65–70°C for 10 min then vortexed. The GC/TPP mixture stands at room temperature for at least 30 min before incorporation of DNA. To prepare DNA-GC/TPP nanoparticles (DNA loading), DNA was loaded into GC/TPP nanoparticles through electrostatic interactions between DNA and GC/TPP nanoparticles by incubating DNA with GC/TPP polymer complex solutions for 20 min. The ratios of GC to plasmid DNA (N/P = w/w) were 3:1, 5:1, 10:1, 20:1, and 30:1 respectively. Finally, appropriate PBS was added into the mixture prior to the transfection assays. The pH of PBS was adjusted respectively according to the pH value of GC/TPP polymer complex solutions. Measurements of Size, morphology and zeta potential of nanoparticles The morphology of the nanoparticles was examined using a transmission electron microscope (TEM: H-7651, Japan) and measured by granule diameter. Appropriate amounts of sample were placed on a copper grill covered with nitrocellulose then dried at room temperature. After negatively staining with tungsten phosphate, they were examined with electron microscopy. The particle size and zeta potential of GC/TPP nanoparticle were measured by dynamic light scattering (DLS) particle size analyzer
J. BIOSCI. BIOENG., (LB550, Horiba Instruments Inc., Horiba, Japan) and a 3000HS/IHPL Zetasizer (Malvern Instruments Ltd., Malvern, UK), respectively. Agarose gel electrophoresis The DNA binding ability of GC/TPP nanoparticles was evaluated by agarose gel electrophoresis. The nanoparticle solutions of pEGFP with GC/TPP polymer were loaded into individual wells of 1% agarose gel and separated at 100 V for 40 min, then stained with 0.5 g/ml ethidium bromide. The resulting plasmid migration pattern was revealed under UV light. Cell Culture and transfection The hepatoma cell line SMMC7721 cells were cultured in RPMI1640 standard growth medium containing 10% fetal bovine serum (FBS) at 37°C under 5% CO2 atmosphere. After the cells reached 50% of confluence, the culture was diluted 1:8 for transfection. Cells were plated at 5 × 104 cells/well containing 500 μl complete medium in clear 24 well tissue plates. After incubation for 24 h, the medium was discarded and the cells were washed once with PBS at the same pH used for transfection as well as with the GC/TPP solution. Nanoparticles containing DNA solution were added to the cells at a concentration of 0.3 μg/well and incubated for 4–5 h. Finally, the solution was discarded and fresh complete medium was added to the cells again. Fluorescene microscopy Fluorescence microscope images were recorded with a DP controller software at 24 and 48 h after transfection. An inverted microscope (IX71, Olympus; Japan) equipped with a high pressure mercury lamp (CST-5000C, Japan) and an appropriate set of filters for fluorescein (FITC) excitation and emission wavelengths were used. Flourescence-activated cell sorting analysis (FACS) After transfection and incubation at 37°C for 48 h, the hepatoma cells were first checked for GFP overexpression by use of fluorescent microscope described above, then the cells were washed with PBS first and followed by a treatment of 0.25% trypsin and 0.02% EDTA (Beyotime, China) until detachment of the cells. Then 1 ml of complete medium was added and the mixture was centrifuged. Finally the cells were washed again with PBS at physiological pH and replenished. Finally, the samples were transferred to FACS tubes and analyzed directly. For each sample, 1 × 106 cells diluted with 300 μl PBS were counted and analyzed with BD FACSDiva software using appropriate controls and gates. Dead cells and cell debris were excluded from the calculation for the percentage of cells expressing GFP. Statistical analysis Each experiment was performed in triplicate and the value refers to the mean ± standard deviation. A SPSS software package (v.16) was used for statistical analyses. For assessment of the statistically significant homogeneous subset, one-way ANOVA tests were performed followed by a Student–Newman–Keuls ranking with a confidence level of α ≤ 0.05.
FIG. 1. The size and zeta potential of GC/TPP nanoparticles at different pH of GC solutions. (A) Effects of the pH of GC solutions on the size of GC/TPP nanoparticles (w/w = 5:1). (B) Comparison of the diameter of GC/TPP and GC/TPP-DNA nanoparticles (N/P = 20:1). (C) Zeta potential of GC/TPP complex. (D) pH of the GC/TPP complex solution.
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TABLE 1. The average diameter and zeta potential of GC-TPP/DNA under various N/P ratios (the pH of GC solution is 6.0 at 25°C). N/P ratio (w/w) 10:1 20:1 30:1
Z-average (nm)
Zeta potential (mV)
279 226 185
11.8 14.4 22.6
RESULTS Characterization of GC/TPP nanoparticles Several factors, such as pH, molecular weight of chitosan and its deacetylation level, all affect the physical and chemical properties of nanoparticles, which further influence their stability, loading ability of DNA and the transfection efficiency of the nanoparticles (19–22). Therefore, we first characterized the physical properties of GC/TPP nanoparticles. As shown in Fig. 1A, an increase of the pH value of the GC solution for particle preparation results in the formation of significantly smaller GC/TPP nanoparticles, which is believed to increase the solubility of particles at physiological pH and improve the efficiency of DNA release (23–25). However when pH exceeded 6.2, GC solution was turbid and could not form nanoparticles with TPP. Loading of pEGFP DNA into the nanoparticles slightly increased the size of particles in contrast to the empty GC/TPP nanoparticles (Fig. 1B). To get an idea about the stability of GC/TPP nanoparticles, the zeta potential, which indicates the present repulsive force and is widely used to predict the long-term stability of the particles, was determined. As shown in Fig. 1C, under all the conditions the nanoparticles show high values of positive zeta potential, which suggest that they tend to repel each other and not come together therefore representing a nice longterm stability under tested conditions. Furthermore, no drastic differences of the zeta potential with various pH ranging from 4.8 to 6.2 were observed. In addition, the pH of polymer complex, as shown in Fig. 1D, ranged from 5.8 to 7.1 according to pH of GC (4.8–6.2) and TPP (8.9) thus further indicates the stable property of GC/TPP nanoparticles. Another parameter, the N/P ratio (i.e., the ratio between cationic polymers and DNA, w/w), is believed to play an important role in affecting the degree of complexation like particle diameter, thus further influencing the transfection efficiency and cytotoxicity of carriers. A low N/P ratio would yield physically unstable particle/DNA complexes which leads to poor transfection efficiency due to low amounts of DNA delivery, while those too stable complexes also result in the same problem if N/P ratio is too high because DNA cannot be released (9,26). As shown in Table 1, with increased N/P ratio, the mean size of the nanoparticles decreased and reaches a minimum value of 185 nm at the N/P ratio of 30:1. Correspondingly, the zeta potential increased with the increase of the N/P ratio, suggesting stable particle/DNA complexes formed. The complex formation of GC/TPP nanoparticle with DNA was further evaluated by gel retardation assay, in which nanoparticle/DNA solutions were electrophoresed on agarose gels and visualized with ethidium bromide (Fig. 2A). Condensed DNA by GC/TPP particles remained at the top of the agarose gel at N/P ratio of 10, 20 and 30 (Fig. 2A, lanes 4–6), suggesting DNA was successfully entrapped into GC/TPP nanoparticles. In contrast, unconjugated DNA (Fig. 2A, lane 1) runs into the gel as well as those with low N/P ratios (Fig. 2A, lanes 2 and 3) due to their unstable states migrate to the positive pole. Fig. 2B shows a TEM photograph of representative GC/TPP-DNA nanoparticles and a spherical and polydisperse nature of the particles is obvious. The particle size distribution was achieved though DLS (LB-550) and was shown in Fig. 3, which indicates that loading of DNA slightly increases the particle size (compare panels A and B in Fig. 3). Transfection studies To test the effectiveness of the prepared GC/TPP-DNA nanoparticles, transfection studies were performed by use of the hepatoma cell line SMMC7721. As shown in Fig. 4, the
FIG. 2. (A) Agarose gel retardation analysis of GC/TPP-DNA nanoparticles. GC/TPP nanoparticles were combined with DNA at different N/P ratios. Lane 1: naked DNA; lane 2: N/P = 3; lane 3: N/P = 5; lane 4: N/P = 10; lane 5: N/P = 20; lane 6: N/P = 30. The gel was 1% and stained with ethidium bromide. (B) TEM photograph of GC/TPP nanoparticles (200 nm).
transfection efficiency varied significantly at different pH values of the cell culture and the highest transfection efficiency was achieved at pH of 6.7 (Fig. 4A). The transfection efficiency varied also at different N/P ratio as analyzed with FACS, and in which, N/P ratio of 20:1 gave the best efficiency compared with 10:1 and 30:1 (Fig. 4B). This result was further confirmed by use of fluorescent microscopy imaging (Fig. 5). A decrease in the transfection efficiency was further observed at other pH values and N/P (data not shown). Most importantly, no transfection was observed when naked DNA was used (Fig. 5A) which further suggests the transfection specificity of the GC/TPP-DNA nanoparticles observed. Furthermore, the pEGFP expression from FACS analysis clearly demonstrated a higher transfection efficiency of the SMMC7721 cell population with GC/ TPP-DNA nanoparticles at the optimal N/P ratio (i.e., 20:1) (with an efficiency of 6.8 ± 0.9%) when compared with the GC/DNA complexes at the same N/P ratio (the efficiency is 0.6 ± 0.2%). Even at nonoptimal N/P ratio of 10:1 and 30:1, the transfection efficiency still reaches 4.6 ± 0.7% and 5.9 ± 0.4%, respectively, which is 7–10 times higher than GC-DNA particles, further illustrating the effective “helper” effect of TPP addition.
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FIG. 4. Optimization of in vitro transfection parameters (0.5 μg of DNA per well and an incubation time of 4 h). (A) The pH effects (at N/P ratio of 20:1). (B) Influence of N/P ratios (the pH of transfection medium was 6.7) and comparison of GC/TPP-DNA, GC-DNA and naked DNA.
FIG. 3. The diameter distributions of nanoparticles observed by DLS (LB550) with pH of GC solution at 6.0. (A) GC/TPP: the mean diameter was 163.5 nm. (B) GC/TPP-DNA: the mean diameter was 198.6 nm, with an N/P ratio of 30:1.
DISCUSSION Due to the abundant expression of asialoglycoprotein (ASGP) in hepatoma cells, drugs or gene carriers conjugated with a ligand has been used to target ASGP receptors for the treatment of liver cancers (13,14). GC is a kind of ASGR based gene carrier to target liver cells. Although several reports already indicate that GC, including other cationic polymers, has the potential to target hepatocytes (27,28), there are few reports thoroughly investigating the transfection efficiency of galactosylated nanoparticles. Furthermore, TPP improves the transfection efficacy of chitosan (16) but has not been tested by use of GC. Therefore, the aim of the present work was to evaluate the conjugation of TPP into GC to test its efficacy as a gene carrier for HepG2 cells targeting. Our results now demonstrate that factors determining a high transfection efficiency depend on the pH of the cell culture, the charge ratio of GC to DNA as well as the preparation means of the nanoparticles. We clearly demonstrate that the size and the zeta potential of GC/ TPP nanoparticles varied in accordance with the pH value of the GC solutions used and an increase of the pH value results in the formation of significantly smaller GC/TPP nanoparticles (Fig. 1). This observation could be explained by the decrease of positive charges of GC while the pH increases, which leads to a reduction of the charge interactions among nanoparticles. After DNA loading, the nanoparticle size increased
slightly. Moreover by complexing GC and TPP, small and stable nanoparticles were achieved leading to an improvement of the transfection efficiency. Furthermore, the characteristics of the nanocarrier system are not the sole determinants of the transfection efficiency since the properties, particularly the pH value, of the cell culture also play an important role (Fig. 4). A higher transfection efficiency was achieved between the pH values of 6.4 and 6.7 and a drastic decrease was observed towards higher pH while a relatively gradual decrease towards the acidic pH, which is correlating with previous reports, i.e. a slightly acidic culture medium is beneficial for a higher transfection efficiency (25,29). This phenomenon is due to the protonation of amine groups of GC under slight acidic conditions which facilitates the binding of negatively charged DNA as well as the DNA condensation into particles through phase separation. However, too acidic pH is not helpful to improve the transfection efficiency because of the strong electrostatic interactions between the negatively charged DNA and the positively charged nanoparticles, which ultimately inhibits DNA release (30). On the other hand, dramatic decrease of the transfection efficiency at higher pH might be a consequence of DNA dissociation from the nanoparticles (31). In addition, the pH of the cell culture should be the same as the GC/TPP polymer complex solutions, otherwise the size of nanoparticles would be changed. Therefore, the composition of the medium should be carefully selected in order to avoid aggregation which ultimately affects the transfection efficiency and the distribution within the cell (22). For example, a simple medium (RPMI1640) or a complete medium (RPMI1640 containing 10% FBS) leads to aggregation of GC/TPP-DNA nanoparticles, forming large particles with diameters ranging from 1 to 2 μm, irrespective of ultrasound dispersion or magnetic stirring
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Furthermore, in vivo or clinical experiments should also be performed to test the system as a novel hepatocyte-targeting gene carrier for its safety, novelty as well as applicability for medical purposes and this work is ongoing in our lab. ACKNOWLEDGMENTS This work was supported by the Foundation of Key project of Sichuan Province (No. 07SG004004). We gratefully acknowledge Prof. Ying Li for kindly providing GC, Prof. Shengfu Li for valuable assistance in microscopic analysis and Dr. Yangyan He for helps in FACS analysis. REFERENCES
FIG. 5. Fluorescent microscopy imaging of transfected cells under optimized parameters. Fluorescent microscopy images were randomly taken from a subset of transfected cells with (A) N:P = 10:1, (B) N:P = 30:1, (C) N:P = 20:1. Transfection experiments were performed in PBS at pH of 6.7, with 0.3 μg per well of pEGFP and incubated for 4 h. Image was taken after 48 h of transfection.
treatment. Of course, no transfection was obtained since there is no cellular uptake of these huge particles. Finally, the N/P ratio is another important factor that affects transfection efficiency (Fig. 4) because a low N/P ratio would yield physically unstable complexes and poor transfection, whereas a high N/P ratio results in a poor transfection because of their stability; thus, the DNA cannot be released from the complexes (1). In conclusion, our results demonstrate that GC, as a derivative of chitosan, has the ability to form stable and small nanoparticles when coupled with TPP at optimal pH solutions due to ionic gelation property of TPP (16). And most importantly, the in vitro analysis using SMMC7721 cells suggests that the GC/TPP nanoparticles have a more effective transfection efficiency when compared with GC itself, thus warranting further improvement of the system. At this point, factors like the degree of deacetylation and molecule weight (MW) of chitosan, pH, serum, charge ratio of chitosan to DNA and cell type and so on all have to be optimized to enhance the transfection efficiency.
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