UNDERSTANDING INTERACTIONS BETWEEN CELLS AND LIPID NANOPARTICLES

了解细胞和脂质纳米粒子之间的相互作用

• 批准号: 8365772
• 负责人: GIULIO CARACCIOLO
• 金额: $ 10.97万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-20 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8365772
• 关键词: Actins; Binding; Cell Culture Techniques; Cell Nucleus; Cells; Cytochalasin D; Cytoplasm; Cytoskeleton; Cytosol; DNA; Data; Diffusion; Equation; Fluorescence; Fluorescent Dyes; Funding; Grant; Image; Label; Laboratories; Lead; Lipids; Liposomes; Microfilaments; Microtubules; Modeling; Motion; National Center for Research Resources; Nocodazole; Pharmaceutical Preparations; Plasmids; Positioning Attribute; Principal Investigator; Reporting; Research; Research Infrastructure; Resources; Role; Signal Transduction; Source; Staining method; Stains; Time; Transfection; United States National Institutes of Health; cost; inhibitor/antagonist; latrunculin B; nanoparticle; particle; plasmid DNA; polymerization; software development; trafficking

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The main aim of the project will be to develop our understanding of the role of the microtubule and actin networks in pDNA transport during liposomal transfection. To elucidate the role of the cytoskeleton networks in the trafficking of plasmids to the cell nucleus, inhibitors of polymerization of actin or microtubule networks will be employed. We will investigate whether disruption of either of these networks lead to the cessation of plasmid transport to the nucleus, a decreased mobility of plasmids, and/or accumulation of plasmid DNA in large aggregates at the cell periphery. Cells will be stained with fluorescent dyes that will bind to the cytoskeleton networks. When in the cytosol unforced diffusion of pDNA-loaded lipid nanoparticles (as detected by co-localization of fluorescence signals arising from fluorescent lipids and labeled DNA) throughout the cell cytoplasm and their spontaneous binding to cytoskeletal networks will be imaged in real time. + Actin filaments. Inhibition of actin polymerization will be achieved by treating cell cultures with latrunculin B or cytochalasin D. + Microtubule network. Nocodazole will disrupt the microtubule network. Experimental strategy. The nanoparticle trajectory (MSD) will be calculated by confocal images as reported previously (Ondrej V. et al., Acta Biochim. Pol. 2007, 54, 657-663). Such strategy will also enable us to track pDNA to assign dynamic parameters to it. The precise role of cytoskeleton networks will therefore be investigated by comparing results (lipid nanoparticle trajectory in the cytoplasm, diffusion parameters of both the pDNA and the MENS nanoparticle) obtained with and without inhibitors of polymerization of actin or microtubule networks. Cytoplasmic transport of plasmid DNA after network disruption using different drug treatments can be also followed. Changes in positions of the fluorescence signals (particle tracking) will be determined using dedicated softwares (developed by Prof. Enrico Gratton), which will allow us to assign 3D coordinates to the fluorescence signals. The diffusion coefficient (D) and the average velocity (v) of the nanoparticles bound to both the networks can be calculated. To quantitatively analyze the motion of the particles, it is helpful to classify the trajectories in terms of simple models. Analytical forms of the equations for various types of motion have been derived (Saxton and Jacobson, Annu. Rev. Biophys. Biomol. Struct. 1997, 26, 373). These models will be fitted to the experimental data, and the best fitting model provides the parameters D, v, and/or, for the particle motion.

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供。子项目的主要支持 并且子项目的主要研究者可能是由其他来源提供的， 包括其他 NIH 来源。 子项目可能列出的总成本 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 该项目的主要目的是加深我们对脂质体转染过程中 pDNA 运输中微管和肌动蛋白网络作用的理解。为了阐明细胞骨架网络在将质粒运输到细胞核中的作用，将使用肌动蛋白或微管网络聚合的抑制剂。我们将研究这些网络中的任何一个的破坏是否会导致质粒向细胞核转运的停止、质粒流动性的降低和/或质粒DNA在细胞外周大量聚集的积累。细胞将被荧光染料染色，荧光染料将与细胞骨架网络结合。当负载 pDNA 的脂质纳米颗粒在细胞质中非受迫扩散（通过荧光脂质和标记 DNA 产生的荧光信号共定位检测到）在整个细胞质中时，它们与细胞骨架网络的自发结合将被实时成像。 + 肌动蛋白丝。通过用 latrunculin B 或细胞松弛素 D 处理细胞培养物可抑制肌动蛋白聚合。 + 微管网络。诺考达唑会破坏微管网络。 实验策略。纳米粒子轨迹（MSD）将通过先前报道的共焦图像来计算（Ondrej V.等人，Acta Biochim.Pol.2007,54,657-663）。这种策略还将使我们能够跟踪 pDNA 并为其分配动态参数。因此，将通过比较使用和不使用肌动蛋白或微管网络聚合抑制剂获得的结果（细胞质中的脂质纳米颗粒轨迹、pDNA 和 MENS 纳米颗粒的扩散参数）来研究细胞骨架网络的精确作用。还可以跟踪使用不同药物处理网络破坏后质粒 DNA 的细胞质转运。荧光信号位置的变化（粒子追踪）将使用专用软件（由 Enrico Gratton 教授开发）确定，这将使我们能够为荧光信号分配 3D 坐标。 可以计算结合到两个网络的纳米颗粒的扩散系数 (D) 和平均速度 (v)。为了定量分析粒子的运动，用简单模型对轨迹进行分类是有帮助的。各种运动类型的方程的解析形式已经被导出（Saxton 和 Jacobson, Annu. Rev. Biophys. Biomol. Struct. 1997, 26, 373）。这些模型将拟合实验数据，最佳拟合模型提供粒子运动的参数 D、v 和/或。