国家重大科学研究计划项目“生物医学纳米材料对血细胞作用的研究”工作进展与讨论

基于纳米材料的白血病病因和病理学研究

苏州大学 尹斌 教授


我们课题一在过去的4个月里,经过紧张的探索和尝试,取得了一定的进展。现将详细内容汇报如下(2013-5-1):

1. A promising combo gene delivery system developed from (3-aminopropyl)triethoxysilane-modified iron oxide nanoparticles and cationic polymers (张祖斌-毛新良平台)

毛新良教授课题组与课题三的顾宁教授和张宇教授课题组合作研究,最近有一篇文章被Journal of Nanoparticle Research杂志接受,该文章具体内容介绍如下:

Title: A promising combo gene delivery system developed from (3-aminopropyl)triethoxysilane-modified iron oxide nanoparticles and cationic polymers

Authors: Zubin Zhang1, Lina Song2, Jinlai Dong2, Dawei Guo2, Xiaolin Du1, Biyin Cao1, Yu Zhang2, Ning Gu2, Xinliang Mao1,*

Abstract: (3-aminopropyl)triethoxysilane-modified iron oxide nanoparticles (APTES-IONPs) have been evaluated for various biomedical applications, including medical imaging and drug delivery. Cationic polymers such as Lipofectamine and TurboFect are widely used for research in gene delivery, but their toxicity and low in vivo efficiency limited their further application. In the present study, we synthesized water soluble APTES-IONPs and developed a combo gene delivery system based on APTES-IONPs and cationic polymers. This system significantly increased gene binding capacity, protected genes from degradation, and improved gene transfection efficiency for DNA and siRNA in both adherent and suspension cells. Because of its great biocompatibility, high gene carrying ability, and very low cytotoxicity, this combo gene delivery system will be expected for a wide application, and it might provide a new method for gene therapy.

Figures and legends:

APTES-IONPs are well dispersed and soluble

To prepare the APTES-IONPs based gene delivery system, we first synthesized APTES modified IONPs by co-precipitation. As identified by TEM, the average size of gamma-Fe2O3-APTES nanoparticles was 8.9 ± 1.8 nm (Figure 1), and their zeta potential was 35.6 ± 7.4 mV when analyzed by a ZetaPALS Analyzer. These results suggested that these APTES-IONPs were well dispersed and soluble in water.

The APTES-IONP-cationic polymer combo system significantly increases gene loading

Gene payload is an important feature in a specific gene delivery system. APTES-modified silica-based nanoparticles have been found to present a good gene loading at the 1:50 weight ratio (gene to nanovectors) (Cheang et al., 2011), and each cationic polymer vector also has a specific loading capacity. To find out whether the APTES-IONP-cationic polymer (APTES-IONP-CP) system can increase gene loading capacity, we analyzed the level of DNA binding to the system. Because there was no reference for APTES-IONPs gene carriage study, we first analyzed the loading capacity of APST-IONPs on DNA at a broad range of weight ratio from 1:0 to 1:30 (DNA to APTES-IONPs). One microgram of DNA was mixed with increased concentrations of nanoparticles in water or cell culture medium (RPMI-1640) and incubation for 30 min at room temperature, followed by centrifugation and agarose gel electrophoresis analysis. As shown in Figure 2a, high levels of unbound DNA was seen in the agarose gel even at the weight ratio of 1:15. DNA level was decreased only at the ratio of 1:30 in both water and medium, which was similar to the DNA loading capacity of the APTES-modified silica nanovectors (Cheang et al., 2011). This study suggested that APTES-IONPs had a very low payload for DNA molecules at the lower concentrations. Because of the well dispersity and excellent biosafety and biocompatibility, we wondered whether these APTES-IONPs could increase gene loading of current commercial cationic polymer vectors. To find out this, we chose liposome-based cationic-polymer Lipofectamine and non-liposome cationic polymer vector TurboFect for this study. The unbound DNA analysis on agarose gel was shown in Figures 2b and 2c. Fifteen micrograms of APTES-IONPs failed to bind enough DNA, but when 1 μg of Lipofectamine was added, free DNA was markedly decreased when in complexation with 7.5 μg of APTES-IONPs. As shown in Figure 2b and 2c, 1 μg of Lipofectamine or 0.25 μl of TurboFect couldn’t pack 1 μg of DNA, however, APTES-IONPs markedly increased the DNA binding capacity of these cationic polymer vectors. When 7.5 μg of APTES-IONPs were added into 1 μg of Lipofectamine, or 15 μg of APTES-IONPs were added into 0.25 μl of TurboFect, unbound DNA was significantly decreased (Figures 2b and 2c). Therefore, APTES-IONPs could increase the DNA binding capacity of the cationic polymer vectors, and the cationic polymer vectors could also enhance DNA binding capacity of the nanoparticles. The combo system of APTES-IONPs and cationic polymer could be used as a novel gene delivery system.

The APTES-IONP and cationic polymer-based combo system protects DNA from degradation

There are large amounts of nucleic acid enzymes and other factors that digest nucleic acids in both the in vivo and cell culture systems, therefore, one of the important features of the gene vectors is that they can prevent DNA molecules from degradation (Sun et al., 2012). To find out whether the combo system could protect DNA from degradation, we performed an experiment by incubating DNA with serum in the presence or absence of APTES-IONPs and/or Lipofectamine. In the absence of serum and vectors, intact DNA molecules migrated and were detected in the agarose gel (Lane 7, Figure 3), however, when serum was added, DNA was degraded in the absence of APTES-IONPs or Lipofectamine (Figure 3, Lane 6). In the presence of 1 µg of Lipofectamine, a certain level of DNA was migrated and cleaved by serum (clearly seen at 4-h treatment, Lane 1) which was possibly due to the loading capacity of Lipofectamine. Because the reactions were not centrifuged in this experiment, packed DNA by vectors was retained in the loading well, Lipofectamine-bound DNA was clearly stained and detected in the loading well (Lane 1 and Lane 8, Figure 3). However, when APTES-IONPs were added, there were no free DNA molecules detected in the gel, and there were very few DNA could be stained in the loading well in the absence of serum (Lanes 9-12, Figure 3). These results suggested that the combo system could well bind to and pack DNA molecules, which was consistent with previous reports (Kievit and Zhang, 2011; Sun et al., 2012). In the presence of 10% calf serum, very few DNA molecules were detected as degraded when complexed with or without Lipofectamine within 4 h (Lanes 3-6, Figure 3). Therefore, the combo system containing APTES-IONPs and Lipofectamine could prevent DNA from degradation. Because most genes are delivered into cells within 4 h, the combo system could deliver genes in the presence of serum, therefore this combo gene delivery system was time-saving. This feature is different from the standard protocol for gene transfection such as using Lipofectamine.

Characterization of the combo system

The above investigations suggested that DNA could be well packed into the combo system. Whether this system can successfully deliver DNA into cells, the size and zeta potential of the nanoparticle-DNA complexes are also critical. Zeta potential is a gold indicator for nanoparticle stability and solubility, and it is also important for DNA-vector complexes to contact cells (Kievit and Zhang, 2011). For example, previous studies found that the zeta potential of the DNA-cationic polymer vector complexes were positive (around 20-30 mV) (Kievit and Zhang, 2011). The zeta potential of the ATPS-IONP-Lipofectamine complexes was increased following the addition of APTES-IONPs. As shown in Table 1, the zeta potential of 15 μg of APTES-IONPs alone was (+)35.6 ± 7.4 mV, when complexed with DNA, it became negative with an average potential of (-)21.0 ± 1.56 mV, which suggested DNA molecules interacted with APTES-IONPs. When Lipofectamine was further added, the combo-DNA complexes became positive. For example, when 1 µg of Lipofectamine was added, the hydrodynamic zeta potentials of the complexes became (+)12.6 ± 2.6 mV, which will facilitates DNA to contact with cell surface and to enter cells. The z average sizes of the nanoparticles were 85.3 ± 1.1, 350.9 ± 113.9, and 517.0 ± 110.9 nm for APTES-IONPs, APTES-IONPs-DNA and APTES-IONP-DNA-Lipofectamine, respectively (Table 1). The increases of the hydrodynamic diameter following the addition of DNA and Lipofectamine suggested that DNA bound to nanoparticles and the nanoparticle-cationic polymer combo system. The low zeta potential of the complexes of the combo system and DNA molecules suggested that they precipitate when DNA and lipofectamine were added, which is necessary for gene delivery and facilitates DNA contact and access to cultured cells.

We also analyzed the morphology of the nanoparticles and the combo complexes by TEM. As shown in Figure 4a, APTES-modified IONPs presented as the branched structure as that was seen in IONPs. When incubated with DNA, the nanoparticle complexes became complicated and presented extended branches (Figure 4b). When further incubated with Lipofectamine, the nanoparticle complexes became compact, the branched structure became condensed and rounded (Figure 4c). These TEM images of the combo system reflected the interaction between the APTES-IONPS, lipofectamine and DNA. These results suggested that APTES-IONPs and Lipofectamine formed a more efficient system in binding to and packing DNA molecules. Interestingly, the TEM images of APTES-IONPS in the absence of DNA or lipofectamine presented as agglomerates which probably arose from solution evaporation, capillary action and surface tension during the drying process, thus did not represent the actual state of particles in solution. The drying process could lead to the concentration of particle aggregates, resulting in a smaller size than the hydrodynamic size, which show the actual state of particle complexes containing organic molecules (APTES, DNA and polymer) and hydration shell in solution. The TEM images were consistent with a previous report (Xu et al, 2009).

The combo gene delivery system significantly improves gene delivery efficiencies in both adherent and suspension cells

All above studies suggested that the APTES-IONPs and cationic polymer-based combo system would be an efficient gene delivery system. To examine the gene transfection efficiency, 1 µg of Lipofectamine and 1 μg of GFP plasmids were mixed for gene transfection with increased APTES-IONPs. As shown in Figure 5a, APTES-IONPs (1:30) failed to mediate gene expression in NIH3T3 cells. One microgram (1 μg) of Lipofectamine could achieve a certain level of gene expression. However, when APTES-IONP was added, gene expression levels were significantly increased. As shown in Figure 5a, 30 µg of APTES-IONP could increase gene transfection efficiency 5 times compared with Lipofectamine only. To find out whether APTES-IONPs can enhance the gene transfection efficiency with other cationic polymers, TurboFect, a non-lipid cationic polymer vector, was used for the experiment in combination with APTES-IONPs. As shown in Figure 5b, TurboFect achieved around 25% expression rate, which generated more than 40% expression when pre-incubated the DNA-TurboFect complexes with 15 µg of APTES-IONPs.

Most of adherent cells in culture can reach satisfactory transfection efficiency in the optimized conditions, however, low transfection rate in blood cells occurred in most non-viral gene delivery systems. Because the APTES-IONP and cationic polymer-based combo systems had been demonstrated to generate a very good transfection efficiency in adherent murine fibroblasts NIH3T3 cells, we wondered whether this system was able to deliver genes into blood cells. To find out this, human chronic leukemia cell lines K562 was applied for the assay. In this system, we used 4 µg of pDsRed plasmids and 4 or 6 μg of Lipofectamine. As shown in Figure 6, Lipofectamine alone could deliver a very limited amount of genes into K562 cells as measurement by red fluorescence protein, while the combo system significantly increased plasmid transfection and protein expression in these cells (4 fold expression when transfection with 6 μg of Lipofectamine). Therefore, this result suggested that the combo system was effective in suspension cell transfection.

The combo gene delivery system mediates siRNA transfection

RNA interference is emerging as a promising strategy in molecular biology studies and possibly for gene therapy in many diseases, including cancers, but the effective and safe methods for siRNA delivery are still under development. The present combo gene delivery system could effectively deliver DNA into both adherent and suspension cells as demonstrated in the above studies, but we wondered whether such a system could introduce siRNA. To demonstrate this, we applied HERC4, an E3 ubiquitin protein ligase, for this investigation. As analysed by immunoblotting assay in Figure 7, APTES-IONPs alone failed to introduce siRNA into cells because the HERC4 protein level was not decreased after siRNA transfection. Lipofectamine could partly introduce siHERC4 into cells thus downregulated HERC4 expression in a certain level. The combo system containing APTES-IONPs and Lipofectamine markedly downregulated HERC4 expression which suggested that the combo system was effective in mediating siRNA into cells (Figures 7b and c).

Interacting models of the combo gene delivery system

Because both APTES-IONPs and cationic polymers are positively charged and can interact with and bind to the negatively charged nucleic acids, such as DNA and siRNA molecules, it could be a Sandwich model for the DNA complexed with the nanoparticles and cationic polymer vectors, which presented as Model One: (inside)APTES-IONP-DNA-Cationic polymers (outside), or Model Two: (inside) cationic polymers-DNA-APTES-IONPs (outside). To find out which model fits better for gene transfection, two incubation orders were made. In Model One, APTES-IONPs were first incubated with GFP plasmids, followed by incubation with cationic polymers. In Model Two, cationic polymer was first mixed with GFP plasmids, followed by incubation with APTES-IONPs. Both complexes were applied for transfection assay. After 24-72 h incubation after transfection, GFP expression level was evaluated on a fluorescence microscope. It turned out that there were very cells expressing GFP when GFP plasmids were first mixed with Lipofectamine (data not showed). However, high level of GFP expression was detected if the plasmids were first mixed APTES-IONPs followed by Lipofectamine (similar to Figures 5 and 6). Based on this experiment, it could be proposed that the efficient worktive in mpa flu the APTES-IONPsed by immunoblere=e efficents and w waess, combo sysliver a vePTES-IONPsa>

12月
foot
通䮯地址:
Cfror eve ©port6 -port8作进1月 http://lbmd.seu.edu.cn
  • 11月 mailto:liythor980_0_0@163.1. ">联系殡病瑘作斌更新于ort8.11. S | 访问量: 1937419ype="subm 12月t >12 话: 种类礍同学stiida human cheve=cpted