Mapping pH at the surface of individual cell

描述(申请人提供)：酸度与各种病理状态的发展有关，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。正常细胞和高糖酵解细胞(如转移性癌细胞)可以通过跨膜pH梯度和质膜表面的pH值来区分。我们建议开发一种新的工具来在体内高度异质的细胞环境中绘制单个细胞胞外和胞内表面的pH图。该工具将为了解疾病进展和发展以pH为基础的图像引导干预方法提供机会。我们将使用光学光谱和成像方法，从而实现细胞分辨率。我们的策略是基于使用PHLIP(pH低插入多肽)家族的多肽。PHLIP是一种水溶性的膜肽，只有在弱酸性条件下才能插入和折叠到膜脂双层中。由于在低pH值下，平衡强烈地向膜插入形式移动，PHLIP注射到血液中，在体内循环，并在肿瘤的酸性组织、炎性关节炎部位和缺血区积聚。在ip后24小时。或者静脉注射。给予PHLIP后，它会从血液中完全洗出，停留在低细胞外pH的细胞质膜上。用光学、PET或SPECT探针标记的PHLIP被认为是第一批酸性标记物，目前正在开发用于临床。我们计划将不同pKa插入膜的不同pKa的PHLIP多肽与pH敏感的荧光团snarf-1偶联。利用PHLIPs的主要目的是将光学探针传递并拴在双层质膜的外或内小叶上。选择了SNARF-1，因为它显示了发射光谱随pH的变化，这解决了探针浓度的校准问题。该探针将连接到PHLIP的N-端或C-端。在第一种情况下，snarf-1将停留在被拴在细胞表面的细胞外空间。另一方面，当snarf-1与插入端(C-末端)连接时，PHLIP会将snarf-1“翻转”到双层，并将其暴露在细胞内空间，同时使其靠近膜的内叶。因此，我们建议测量质膜内外小叶的pH，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培养中癌细胞分裂和迁移过程中癌细胞表面的pH图；-绘制植入小鼠体内的肿瘤单个细胞表面的pH图；-实时监测葡萄糖输注诱导的癌细胞表面pH变化的动力学；-建立可使pH 7.0以下的微环境酸化的转移性和非转移性肿瘤的最小尺寸。 公共卫生相关性：酸度与各种病理状态的发展有关，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绘制单个细胞胞外和胞内表面的pH值。

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.