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。通过跨膜pH梯度和质膜表面pH值可将正常细胞与高度糖酵解的细胞（如转移性癌细胞）区分开来。我们建议开发一种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。该工具将为了解疾病进展和开发基于ph的图像引导干预方法提供机会。我们将采用光谱学和成像方法，从而实现细胞分辨率。我们的策略是基于pHLIP （pH低插入肽）家族的肽的使用。philips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分子层中。由于在低pH下平衡强烈地向膜插入形式转移，因此pHLIP注入血液，在体内循环，并在肿瘤、炎性关节炎部位和缺血区域的酸性组织中积累。在口服或静脉注射pHLIP 24小时后，它被完全从血液中冲洗出来，停留在细胞外ph值较低的细胞的质膜中。用光学、PET或SPECT探针标记的pHLIP被认为是第一个酸度标记物，目前正在开发用于临床应用。我们计划用ph敏感荧光团snif -1将不同pKa的phillip肽偶联到4.5 - 6.5的膜上。使用philips的主要目的是将光学探针传送和系留到质膜双分子层的外层或内层。选择SNARF-1，因为它显示了发射光谱随pH值的变化，解决了探针浓度的校准问题。探针将连接到philips的N-或C-端。在第一种情况下，snaff -1将停留在细胞外空间，被拴在细胞表面。另一方面，当snawf -1与肽插入端（c端）结合时，pHLIP会将snawf -1“翻转”穿过双分子层，使其暴露在细胞内空间，同时使其靠近膜的内小叶。因此，我们建议从质膜的内外小叶测量pH值，并确定跨膜的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：-在三维培养中绘制细胞分裂和迁移过程中癌细胞表面的pH值；-绘制植入小鼠体内的肿瘤中单个细胞表面的pH值；-实时监测葡萄糖输注诱导的癌细胞表面pH变化动力学；-确定转移性和非转移性肿瘤的最小尺寸，可以酸化pH 7.0以下的微环境。

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.