壳聚糖基因纳米粒子的细胞摄入和体外毒性研究

用分子量为50kDa和400kDa的壳聚糖分别和[α-32P]dATP标记的质粒DNA,在不同的N/P比下,通过复凝聚方法形成基因纳米粒子。对形成的壳聚糖基因纳米粒子(Chitosan gene nanoparticle,CGN)进行表征,评价CGN体外细胞毒性,研究两种壳聚糖形成的基因纳米粒子被A10和K562细胞摄入的量和速度。结果表明:(1)随着壳聚糖分子量和N/P比的增大,形成的基因纳米粒子更易于进入细胞,同时显示纳米粒子的zeta电位与细胞摄入量之间存在关联性;(2)壳聚糖基因纳米粒子的毒性远远小于商品化的细胞转染试剂Lipofectamine2000。

壳聚糖基因纳米粒子对L02细胞的作用

目的研究壳聚糖基因纳米粒子(CNP)、精氨酸化壳聚糖基因纳米粒子(ANP)和十六烷基化壳聚糖基因纳米粒子(HNP)对人正常肝细胞系L02细胞的作用。方法采用复凝聚方法制备CNP、ANP和HNP,粒度及zeta电位分析仪进行纳米粒子表征。将浓度分别为5、10、30、50μg/ml(以DNA含量计)的各种纳米粒子与L02细胞共育,采用MTT法进行细胞毒性评价,流式细胞分析技术进行细胞凋亡检测。结果CNP、ANP和HNP的zeta电位为12.10～14.63mV,粒径约为148.07～179.47nm。当各基因纳米粒子浓度为30μg/ml时,细胞存活率开始降低;达到50μg/ml时,存活率降至最低。当各基因纳米粒子浓度为30μg/ml时,可以诱导L02细胞发生凋亡,随纳米粒子浓度的升高,凋亡率升高。结论当达到一定浓度时,CNP、ANP和HNP对L02具有一定细胞毒性,可诱导细胞产生凋亡。

精氨酸修饰壳聚糖基因纳米粒子对血液补体活性和溶血作用的影响

目的 考察精氨酸修饰壳聚糖基因纳米粒子（ACGN）对血液补体活性和溶血的影响.方法 将精氨酸接枝到壳聚糖上制备精氨酸修饰的壳聚糖,用共沉降的方法制备精氨酸修饰壳聚糖基因纳米粒子,用光子相关光谱检测精氨酸修饰壳聚糖基因纳米粒子的粒径,用凝胶电泳阻滞实验对精氨酸修饰壳聚糖与DNA的相互作用进行研究,并考察壳聚糖基因纳米粒子（CGN）和ACGN在体内外对血液补体活性和溶血作用的影响.结果 精氨酸修饰壳聚糖在电荷比为2∶1时能有效地完全包覆DNA,并形成粒径大约为120～180nm的纳米粒子,ACGN和CGN在体内外对红细胞没有明显的破坏作用,在体外对补体系统均没有明显的影响作用,但在体内引起补体的轻微升高.结论 ACGN在体内外没有引起明显的补体激活和溶血作用,有望成为一种新型的、安全的非病毒基因载体.

精氨酸修饰壳聚糖基因纳米粒子对血小板GMP-140表达的影响

目的 考察精氨酸修饰壳聚糖基因纳米粒子（ACGN）对血小板GMP-140表达的影响.方法 制备精氨酸修饰的壳聚精（ACS）并朋红外光谱对其进行表征;用共沉降的方法制备精氨酸修饰壳聚糖基因纳米粒子,用凝胶电泳阻滞实验对精氨酸修饰壳聚精与DNA的相互作用进行研究;用酶联免疫双抗夹心法测定血小板α颗粒膜蛋白-140（GMP-140）表达来考察精氨酸修饰壳聚糖基因纳米粒子对血小板激活的影响.结果 红外光谱结果显示精氨酸成功地接枝到壳聚糖上,在正负电荷比≥2：1时,ACS能完全阻滞DNA的迁移,表明所有的DNA均已被ACS完全包覆.壳聚糖基因纳米粒子（CGN）和ACGN在体外均不引起血小板的激活,但在体内则都引起轻微血小板激活.结论 精氨酸修饰壳聚糖纳米粒子对血小板GMP-140的表达没有引起明显的变化,有望成为一种新型的、安全的非病毒基因载体.

壳聚糖基因纳米粒子诱导L02细胞凋亡的机制研究

目的研究壳聚糖基因纳米粒子（CGNP）引起人正常肝细胞系L02细胞凋亡的分子机制。方法通过复凝聚方法制备CGNP，用原子力显微镜和粒度分析仪进行纳米粒子的表征；将浓度分别为5，10，30，50ug／mL（以DNA含量计）的CGNP与L02细胞共育，采用荧光分析技术检测活性氧自由基（ROS）的含量，运用Western-blot方法检测细胞色素C（Cyt C）的释放。结果CGNP平均粒径为115．6nm，粒径范围在74．14～136．28nm。当CGNP的浓度大于30ug／mL时，胞浆中有少量的Cyt C释放；当浓度大于50ug／mL时，ROS的量显著增加。结论当达到一定浓度时，CGNP可影响细胞线粒体的功能，从而引起细胞凋亡。

水/油/水双乳化技术制备高效性神经生长因子

背景:神经生长因子是大分子蛋白类物质,很难透过血脑屏障,基因治疗可以有效地将神经营养因子投递到脑内。目的:探索制备高效性神经生长因子基因纳米粒子的方法。方法:利用水/油/水(W/O/W)双乳化技术制备神经生长因子基因纳米粒子,用壳聚糖对其进行表面修饰,同时偶联Tat多肽。切断穹隆-海马伞诱使基底前脑胆碱能神经元溃变。16只SD雄性大鼠随机数字表均分成损伤组和基底前脑显微注射神经生长因子基因的纳米粒子组。结果与结论:制备的纳米粒子粒径分布为110～150nm。壳聚糖可明显提高其表面正电荷和包封率,但对其粒径大小影响不大。偶联Tat多肽则明显提高其转染效率。说明利用水/油/水技术可以成功制备神经生长因子基因纳米粒子。该粒子移植后对基底前脑有明显的保护作用。

Magnetic iron oxide nanoparticles carrying PTEN gene to reverse cisplatin-resistance of A549/CDDP cell lines

To evaluate the feasibility of using magnetic iron oxide nanoparticle as wild PTEN gene carrier for transfection in vitro to reverse cisplatin-resistance of A549/CDDP cells, A549/CDDP cells were transfected with the wild PTEN gene expression plasmid (pGFP-PTEN) by magnetic iron nanoparticle and lipo2000. The transfection efficiency was detected by fluorescence microscope and flow cytometer. The expression levels of PTEN mRNA and protein were detected by reverse transcription polymerase chain reaction (RT-PCR) and immunocytochemistry analysis. The effect of PTEN transfection on cell cycle enhances the sensitivity of A549/CDDP to cisplatin and nanoparticle-mediated transfection has a higher efficiency than that of the liposome-mediated group. The apoptosis level was up-regulated in PTEN transfection group. The magnetic iron oxide nanoparticle could be used as one of the ideal gene carriers for PTEN gene delivery in vitro. PTEN can be an effective target for reversing cisplatin-resistance in lung cancer.

NANOPARTICLE AS A NEW GENE TRANSFERRING VECTOR IN SPECIFIC EXPRESSION GENE

Objective. To evaluate the possibility and efficiency of nanoparticle as a new vector in specific gene transference.Methods. Nanoparticle-DNA complex was prepared with Poly- dl-lactic-co-glycolic acid (PLGA) bearing anti-sense monocyte chemotactic protein-1 (A-MCP-l), a specific expression gene, and the package efficiency, release progress in vitro, and the size of the complex were determined. The possibility of the new vector was evaluated with genomic DNA PCR by transferring gene into cultured smooth muscle cells (SMC), cationic lipids as a control. For study in vivo, jugular vein-to-artery bypass grafting procedures were performed on 20 New Zealand white rabbits, of which 6 grafts were transferred with nanoparticle-A-MCP-1 (200 μg), 6 with A - MCP-1 (200 μ g) by cationic liposome, 4 with LNCX plasmid, and 4 as control. Fourteen days after the grafts were harvested, the expression of A-MCP-l and its effect on MCP-1 in vein grafts were detected by dot blot, and the morphologic evaluation of grafts was performed.Results. The package efficiency of the nanoparticle-DNA complex was 0. 9% , release progress in vitro lasted 2 weeks, and the size ranged from 150 to 300nm. SMC genomic DNA PCR showed that A-MCP-l gene could be successfully transfected into cells by nanoparticle. The study in vivo indicated that A-MCP-l mRNA was expressed in both local gene delivery groups, nanoparticle and liposome, meanwhile, MCP-1 expression in vein grafts was significantly inhibited and neointimal hyperplasia was notably reduced.Conclusion. Nanoparticle can act as a vector to transfect specific gene.

Gold nanoparticle-based anodic stripping voltammetry detection of transcription factor NF-kB

Transcription factor and sequence specific DNA interactions play important roles in drug genome and transcription diagnosis. Gold nanoparticles show high sensitivity, stability and compatibility for biological molecules as electrochemical intercalators. Here unimolecular hairpin oligonucleotides were self-assembled onto Au electrode surface and elongation on solid phase was carried out to double strand oligonucleotides with transcription factor NF-r,13 binding site. Gold nanoparticle-catalyzed Ag deposition was detected by anodic stripping voltammetry （ASV） for NF-kB binding. It was indicated that this method for sequence specific DNA binding protein detection shows pronounced specificity, sensitivity and we can find application in transcription regulation research, open reading frame characterization and functional gene inspection by this method.

壳聚糖作为基因输送载体的最新研究进展

基因治疗是一种非常有前景的医学治疗技术，然而目前尚不能广泛地应用于临床，安全高效的基因输送载体的缺乏是其临床推广应用的主要限制因素之一。壳聚糖作为一种天然存在的阳离子多聚物，具有较好的生物相容性和生物降解性，可以与DNA通过快速混合形成纳米颗粒，从而具有作为基因输送载体的重大潜力。就壳聚糖作为基因输送载体的最新研究进展进行系统综述，重点讨论纳米粒子进入体内后需要跨越的各个生物屏障以及如何克服这些生物屏障。

Prussian blue nanoparticle-loaded microbubbles for photothermally enhanced gene delivery through ultrasound-targeted microbubble destruction

By adsorbing chitosan(CS)-functionalized Prussian blue(PB) nanoparticles(CS/PB NPs) complexing DNA onto the surface of gas encapsulated microbubbles(MBs), a multifunctional gene delivery system of MBs@CS/PB/DNA was fabricated for photothermally enhanced gene transfection through ultrasound-targeted microbubble destruction. CS/PB NPs of(2.69 ± 0.49) nm could complex DNA effectively when the mass ratio was2:1. It was found that MBs@CS/PB/DNA could enhance ultrasound imaging greatly both in vitro and in vivo. In addition, MBs@CS/PB/DNA could be disrupted by applying a higher-intensity ultrasound irradiation to release CS/PB/DNA, which could effectively transform the nearinfrared(NIR) light into heat to assist the uptake of CS/PB/DNA by cells. With the aid of ultrasound irradiation and NIR light irradiation, the gene transfection efficiency was significantly enhanced to(43.08 ± 1.13) %, much higher than polyethylenimine. Moreover, MBs@CS/PB/DNA showed excellent biocompatibility, encouraging the further exploration of MBs@CS/PB/DNA to be a platform for combined ultrasound image, photothermal therapy, drug delivery, and gene therapy.

Engineering imaging probes and molecular machines for nanomedicine

Nanomedicine is an emerging field that integrates nanotechnology, biomolecular engineering, life sciences and medicine; it is expected to produce major breakthroughs in medical diagnostics and therapeutics. Due to the size-compatibility of nano-scale structures and devices with proteins and nucleic acids, the design, synthesis and application of nanoprobes, nanocarders and nanomachines provide unprecedented opportunities for achieving a better control of biological processes, and drastic im- provements in disease detection, therapy, and prevention. Recent advances in nanomedicine include the development of func- tional nanoparticle based molecular imaging probes, nano-structured materials as drug/gene carders for in vivo delivery, and engineered molecular machines for treating single-gene disorders. This review focuses on the development of molecular imag- ing probes and engineered nucleases for nanomedicine, including quantum dot bioconjugates, quantum dot-fluorescent protein FRET probes, molecular beacons, magnetic and gold nanoparticle based imaging contrast agents, and the design and validation of zinc finger nucleases （ZFNs） and TAL effector nucleases （TALENs） for gene targeting. The challenges in translating nano- medicine approaches to clinical applications are discussed.

Biomimetic gold nanocomplexes for gene knockdown： Will gold deliver dividends for small interfering RNA nanomedicines？

RNA interference （RNAi） effectors such as small interfering RNA （siRNA） and micro RNA （miRNA） can selectively downregulate any gene implicated in the pathology of a disease. Therefore, RNAi-based therapies have immense potential for the treatment of a wide range of diseases. However, pharmacokinetic and pharmacodynamic studies have revealed that these therapeutic agents have poor bioactivity due to a number of factors, including insufficient plasma drug levels, short plasma half-lives, renal clearance, and hepatic metabolism. Non-viral delivery may facilitate the clinical application of siRNA-based therapeutics by helping to overcome these barriers. Recently, the potential of gold nanoparticles （AuNPs） as multifunctional carriers for transporting drugs, proteins, and genetic materials has been demonstrated. In this review, some of the key properties of AuNPs relevant to siRNA delivery, such as physical properties and surface chemistry have been described. In addition, the ability of AuNP-based formulation strategies to successfully overcome delivery barriers associated with siRNA, and the potential for this material to translate into safe and effective nanomedicines are critically discussed.