Mapping pH at the surface of individual cell

描述（由申请人提供）：酸度与各种病理状态的发展相关，如实体瘤、缺血性中风、神经创伤、癫痫发作、炎症、感染、伤口、囊性纤维化等。正常细胞可以通过跨膜pH梯度和质膜表面的pH值来区分与高度糖酵解的细胞（例如转移性癌细胞）。我们建议开发一种新的工具来映射在细胞的细胞外和细胞内表面的pH值在体内细胞的高度异质性的环境。该工具将允许打开一个机会，有助于了解疾病的进展和基于pH值的图像引导干预方法的发展。我们将采用光学光谱和成像方法，这使得实现细胞分辨率。我们的策略是基于使用pHLIP（pH低插入肽）家族的肽。pHLIPs是一种水溶性的膜肽，仅在微酸性条件下插入并折叠在膜的脂双层中。由于在低pH下平衡强烈地向膜插入形式移动，pHLIP被注射到血液中，在体内循环并在肿瘤的酸性组织、炎性关节炎的部位和缺血区域中积累。在腹膜内或静脉内给予pHLIP后24小时，其完全从血液中洗出并停留在具有低细胞外pH的细胞的质膜中。用光学、PET或SPECT探针标记的pHLIP被认为是第一酸度标记物，其目前正在开发用于临床用途。我们计划将插入膜的不同pKa的pHLIP肽与pH敏感的荧光团SNARF-1缀合，pKa范围为4.5至6.5。使用pHLIPs的主要目的是将光学探针递送并拴系到质膜双层的外叶或内叶。选择SNARF-1，因为其显示发射光谱响应于pH的偏移，这解决了探针浓度的校准问题。探针将连接到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中，被拴系到细胞表面。另一方面，当SNARF-1与肽插入端（C-末端）缀合时，pHLIP将使SNARF-1“翻转”穿过双层并将其暴露于细胞内空间，同时使其保持靠近膜的内小叶。因此，我们建议从质膜的外部和内部小叶测量pH值，并确定跨膜pH梯度。将进行溶液，2D和3D细胞培养以及小鼠癌症模型的实验。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; -绘制植入小鼠的肿瘤中单个细胞表面的pH; -监测葡萄糖输注诱导的癌细胞表面真实的pH变化动力学;- 建立转移性和非转移性肿瘤的最小尺寸，其可以酸化低于pH 7.0的微环境。 公共卫生相关性：酸度与各种病理状态的发展相关，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的工具来映射单个细胞的细胞外和细胞内表面的pH。

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.