苏州大学 尹斌 教授
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 working model for the combo gene delivery could be (inside)-APTES-IONP-DNA-Cationic polymers (outside), as showed in Figure 8. This sandwich packing model also explains that DNA packed by this combo system was less detected in the agarose gel analysis as shown in Lanes 2-4, 9-12, Figure 3.
研究表明murine leukemia virus (MuLV) 多效逆转录病毒，可以诱导淋巴性(T- and B-cell)和非淋巴性白血病(myeloid, erythroid, megakaryocytic)。近交系小鼠对白血病因子较敏感，可以作为逆转录病毒诱导的白血病模型。也有研究表明碳纳米管（CNT）具有免疫调节和载体双重性质，激发机体的免疫反应；碳纳米管提升树突细胞对肿瘤抗原的摄取，并增进淋巴细胞对肿瘤细胞的杀伤力。那么，当碳纳米管进入白血病患者体内，是否会与血液细胞相互作用，进而改变恶性肿瘤的进程或状态，有待于深入探索。本实验旨在通过Mol4070LTR病毒感染方法，建立小鼠白血病模型，研究MuLV的致病机制，并初步探索碳纳米管在疾病进程中的影响作用。初步结果如下：
本研究选用某品系小鼠，设置对照组、RV组、CNT组及RV+CNT组，在出生24 h内，RV组及RV+CNT组小鼠腹腔注射浓缩的Mol4070LTR病毒；然后在Week2,4,6,8,10,12,14,16，CNT组及RV+CNT组皮下注射0.2 mg CNT，并持续观察疾病进程。结果发现， 该品系小鼠有明显的发病症状。如小鼠存活曲线图所示（图1）：RV+CNT组较RV组小鼠发病迅速，在4个月的时候已经全部死亡，差别显著；CNT组未发病。
将对照组（con），RV组及RV+CNT组小鼠骨髓（BM）及淋巴结（LN）中的活细胞，进行淋系marker TCR-β及B220染色，及髓系marker CD11b及Gr-1染色，结果显示，RV及RV+CNT组小鼠或者LN中髓系标志表达增加，或者BM中淋系标志表达增加（图4），表明MuLV可诱导小鼠发生髓系/淋系白血病。
本实验涉及的人急性髓系白血病（AML）细胞系有A, B,C, D, E, F，用MTT实验的方法测定不同浓度的纳米银对AML细胞的存活率的影响。图1所示为实验中用到的纳米银的表征。如图2所示纳米银可降低AML细胞的存活率，对其具有杀伤作用，并且浓度越大，杀伤效果越明显；且杀伤作用强度与细胞种类有关，F细胞明显比A细胞敏感。
图1.Ag-NPs的透射电子显微镜图 (A)Ag-NP-PVP 5nm, (B)Ag-NP-PVP 10nm, (C)Ag-NP-PVP 30nm, (D)Ag-NP-PVA 30nm.
图2.经纳米银处理后的白血病细胞系细胞活力的变化。白血病细胞系（A，B，C，D，E与F）经纳米银(PVP, 5nm)处理72小时后，用MTT实验法测定细胞活力。数据代表三个独立实验的平均值±SD表示。*表示p < 0.05，与对照组比较。
图3.不同浓度与不同粒径纳米银(PVP, 5,10 and 30 nm)处理72小时后F细胞活力的变化。用MTT分析实验测定细胞活力. 数据代表三个独立实验的平均值±SD表示。*ap< 0.05 表示与对照组比较, *abp<0.05 表示与5nm粒径Ag-NPs-PVP实验组比较, *abcp<0.05 与5nm粒径Ag-NPs-PVP实验组比较。
图4.包被材料不同但粒径相同的纳米银(PVP和PVA, 30 nm)处理72小时后F细胞活力的变化。用MTT分析实验测定细胞活力。数据代表三个独立实验的平均值±SD表示。*p < 0.05 表示与对照组比较。
图5.纳米银对A与F细胞凋亡的作用。用1.5μg/mL纳米银(5nm, PVP)处理A与F细胞24小时后，采用流式细胞技术检测细胞凋亡的情况，AnnexinV-FITC标记凋亡早期的细胞，PI标记凋亡晚期的细胞。数据代表三个独立实验的平均值±SD表示。*表示实验组与对照组相比p < 0.05。
图6. 纳米银对AML细胞周期的作用。用1.5μg/mL纳米银(5nm, PVP)处理F（A）与A（B）细胞24小时后，采用流式细胞技术检测细胞周期分布的情况，数据代表三个独立实验的平均值±SD表示。*表示p < 0.05。
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