Mapping pH at the surface of individual cell

绘制单个细胞表面的 pH 值

• 批准号: 8413926
• 负责人: Oleg A Andreev
• 金额: $ 19.14万
• 依托单位: UNIVERSITY OF RHODE ISLAND
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-19 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8413926
• 关键词: Acidity; Animal Model; Bathing; Blood; Calibration; Cancer Model; Cell Culture System; Cell Culture Techniques; Cell Division Process; Cell Line; Cell membrane; Cell surface; Cells; Cellular Membrane; Clinical; Cystic Fibrosis; Development; Diagnostic; Disease Progression; Disseminated Malignant Neoplasm; Ensure; Environment; Epilepsy; Equilibrium; Extracellular Fluid; Extracellular Space; Family; Glucose; Goals; Human; Implant; Individual; Inflammation; Infusion procedures; Intracellular Space; Ischemia; Ischemic Stroke; Kinetics; Label; Lipid Bilayers; Liposomes; Maps; Measurement; Measures; Membrane; Monitor; Mus; Neoplasm Metastasis; Normal Cell; Optics; Parents; Peptides; Plasma; Positron-Emission Tomography; Problem Solving; Resolution; Rheumatoid Arthritis; Site; Solid Neoplasm; Solutions; Staging; Stroke; Surface; Time; Tumor Tissue; Water; Wound Infection; base; cancer cell; cell motility; extracellular; fluorophore; image guided intervention; in vivo; neoplastic cell; new technology; novel; pH gradient; research study; response; seminaphthorhodaminefluoride; single photon emission computed tomography; spatiotemporal; spectroscopic imaging; tool; tumor

DESCRIPTION (provided by applicant): The acidity is associated with development of various pathological states such as solid tumors, ischemic stroke, neurotrauma, epileptic seizure, inflammation, infection, wounds, cystic fibrosis and others. Normal cell could be distinguished from highly glycolytic cell (for example, metastatic cancer cell) by transmembrane pH gradient and value of pH at surface of plasma membrane. We propose to develop novel tool to map pH at the extracellular and intracellular surfaces of individual cell in highly heterogeneous environment of cells in vivo. The tool would allow opening an opportunity to contribute in understanding of diseases progression and development of approaches of pH-based image-guided intervention. We will employ optical spectroscopic and imaging approaches, which allow achieving cellular resolution. Our strategy is based on use of peptides of pHLIP (pH Low Insertion Peptide) family. pHLIPs are water-soluble membrane peptides, which insert and fold in lipid bilayer of membrane only at slightly acidic conditions. Since the equilibrium is strongly shifted toward membrane inserted form at low pH, pHLIP injected into blood, circulates in body and accumulates in acidic tissue of tumors, site of inflammatory arthritis and ischemic regions. At 24 h after i.p. or i.v. administration of pHLIP, it is washed out completely from the blood and stays in plasma membrane of cells with low extracellular pH. pHLIP labeled with optical, PET or SPECT probes is considered to be first acidity markers, which are currently under development for clinical uses. We plan to conjugate pHLIP peptides of different pKa of insertion into membrane ranging from 4.5 to 6.5 with pH-sensitive fluorophore, SNARF-1. The main goal of using pHLIPs is to deliver and tether optical probe to the outer or inner leaflet of bilayer of plasma membrane. The SNARF-1 was selected, since it demonstrates shift of the emission spectra in response to pH, which solves the problem of calibration for the probe concentration. The probe will be attached to the N- or C- terminus of pHLIPs. In first case, SNARF-1 will stay in the extracellular space being tethered to the cell surface. On the other hand, when SNARF-1 would be conjugated with the peptide inserting end (C-terminus), pHLIP would "flip" SNARF-1 across the bilayer and expose it to the intracellular space, while keeping it close to the inner leaflet of membrane. Thus, we propose to measure pH from the outer and inner leaflets of plasma membrane and identify transmembrane pH gradient. Experiments in solution, 2D and 3D cell culture, as well as on mouse cancer models will be performed. Our goals are: - to map pH at the surface of cancer cells in a process of cell division and migration in 3D culture; - to map pH on the surface of individual cells in tumors implanted into mice; - to monitor kinetics of pH changes at the surface of cancer cells in real time induced by the glucose infusion; - to establish the minimal size of metastatic and non-metastatic tumors, which can acidify microenvironment below pH 7.0. PUBLIC HEALTH RELEVANCE: The acidity is associated with development of various pathological states such as solid tumors, ischemia, stroke, inflammation, infection, wounds, cystic fibrosis and others. We propose to develop a novel tool to map pH at the extracellular and intracellular surfaces of individual cell.

描述（申请人提供）：酸度与各种病理状态的发展相关，例如实体瘤、缺血性中风、神经外伤、癫痫发作、炎症、感染、伤口、囊性纤维化等。通过跨膜 pH 梯度和质膜表面的 pH 值，可以将正常细胞与高度糖酵解的细胞（例如转移性癌细胞）区分开来。我们建议开发新的工具来绘制体内细胞高度异质环境中单个细胞的细胞外和细胞内表面的 pH 值。该工具将为理解疾病进展和开发基于 pH 值的图像引导干预方法提供机会。我们将采用光学光谱和成像方法来实现细胞分辨率。我们的策略基于 pHLIP（pH 低插入肽）家族肽的使用。 pHLIP 是水溶性膜肽，仅在微酸性条件下插入并折叠在膜的脂质双层中。由于在低pH值下平衡强烈地向膜插入形式转变，pHLIP被注射到血液中，在体内循环并在肿瘤的酸性组织、炎症性关节炎部位和缺血区域中积累。腹腔注射后 24 小时或静脉注射施用 pHLIP 后，它会从血液中完全洗掉，并保留在细胞外 pH 值较低的细胞质膜上。用光学、PET 或 SPECT 探针标记的 pHLIP 被认为是第一个酸度标记物，目前正在开发用于临床用途。我们计划将插入膜的不同 pKa 范围为 4.5 至 6.5 的 pHLIP 肽与 pH 敏感荧光团 SNARF-1 结合。使用 pHLIP 的主要目标是将光学探针传递并束缚到质膜双层的外层或内层。选择 SNARF-1 是因为它展示了发射光谱随 pH 值的变化，这解决了探针浓度的校准问题。探针将连接至 pHLIP 的 N 或 C 末端。在第一种情况下，SNARF-1 将停留在细胞外空间并与细胞表面相连。另一方面，当 SNARF-1 与肽插入端（C 端）缀合时，pHLIP 会“翻转”SNARF-1 穿过双层并将其暴露于细胞内空间，同时保持其靠近内部小叶膜。因此，我们建议测量质膜外叶和内叶的 pH 值并确定跨膜 pH 梯度。将在溶液、2D 和 3D 细胞培养以及小鼠癌症模型上进行实验。我们的目标是： - 绘制 3D 培养中细胞分裂和迁移过程中癌细胞表面的 pH 值图； - 绘制植入小鼠体内的肿瘤中单个细胞表面的 pH 值图； - 实时监测葡萄糖输注引起的癌细胞表面pH变化的动力学； - 确定转移性和非转移性肿瘤的最小尺寸，可以将微环境酸化至pH 7.0以下。 公共健康相关性：酸性与各种病理状态的发展有关，例如实体瘤、缺血、中风、炎症、感染、伤口、囊性纤维化等。我们建议开发一种新工具来绘制单个细胞的细胞外和细胞内表面的 pH 值。