Computationally Guided Approach to Produce Ratiometric Probes Operating in the Red to Near-infrared Region to Accurately Determine pH Levels within Organelles

计算引导方法生产在红色至近红外区域运行的比率探针，以准确确定细胞器内的 pH 水平

• 批准号: 10796036
• 负责人: Rudy Lin Luck
• 金额: $ 46.95万
• 依托单位: MICHIGAN TECHNOLOGICAL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-18 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10796036
• 关键词: A549; Adenocarcinoma; Amino Acids; Anions; Area; BODIPY; Biological; Breast Cancer Cell; Calibration; Cell membrane; Cell physiology; Cells; Cellular Stress; Detection; Dyes; Elements; Endoplasmic Reticulum; Environment; Fluorescence; Fluorescent Probes; Golgi Apparatus; Hela Cells; Human; Hypoxia; Image; Ions; Learning; Link; Lysosomes; MCF7 cell; MDA MB 231; Malignant neoplasm of prostate; Measurement; Metals; Methods; Mitochondria; Molecular; Monitor; Near-infrared optical imaging; Nitrogen; Nutrient; Oligonucleotides; Optics; Organelles; Oxidative Stress; Oxygen; PC3 cell line; Pathologic Processes; Penetration; Permeability; Pharmacotherapy; Physiological Processes; Process; Property; Research; Specificity; Spironolactone; Starvation; Structure; Sulfonamides; Sulfur; Tissues; Toluene; Toxic effect; Xanthenes; absorption; amphiphilicity; benzenesulfonamide; biomaterial compatibility; cell injury; design; ethylene glycol; experimental study; extracellular; fluorophore; insight; light scattering; lung Carcinoma; morpholine; novel; optical spectra; programs; quantum; ratiometric; response; skills; undergraduate student; water solubility

Abstract Employing a computationally guided approach, this project will develop ratiometric fluorescent probes operating in the red to the near-infrared region to accurately determine extracellular and intracellular pH changes across cell membranes and in different organelles, such as lysosomes, mitochondria, the endoplasmic reticulum, the Golgi apparatus, and to monitor mitochondrial delivery to the lysosomes during mitophagy caused by nutrient starvation, hypoxia, and drug treatment. Spectrochemical attributes of probes will be determined before synthesis in order to judge applicability in the desired spectral range. Probes will consist of near-infrared fluorophores linked into near-infrared rhodol dyes to achieve ratiometric responses to pH changes based on pH modulation resulting in π-conjugation changes of the probes. Red to near-infrared fluorophores will include BODIPY dyes, traditional near-infrared xanthene dyes, and their derivatives, where the central oxygen atom of xanthene cores will be replaced by other elements such as Si, S, and N. Spirolactone and spirolactam molecular switches that can be tuned to appropriate pKa values matching the different organelles under study will be used. Specific targeting to different organelles and cell membranes will be achieved by introducing lysosome-specific morpholine, mitochondria-specific triphenylphosphonium, Golgi apparatus-specific benzenesulfonamide, endoplasmic reticulum-specific p-toluene sulfonamide, or cell membrane-specific amphilphilic zwitterion residues through oligo(ethylene glycol) spacers to the fluorescent probes. The ratiometric red to near-infrared fluorescent probes based on spirolactam switches will have pKa values from 4.5 to 6.2 and will only show fluorescence of near-infrared fluorophores under neutral and basic pH conditions since rhodol moieties have closed spirolactam ring structures under the slightly basic conditions within mitochondria. A lysosomal acidic environment will trigger the opening of the spirolactam switches on the rhodol moieties, significantly enhancing π-conjugation between the fluorophore and rhodol moieties, resulting in ratiometric fluorescence responses to pH changes. These processes will also be delineated via theoretical calculations before syntheses are conducted. The probes will have the combined advantages of near- infrared imaging (including deep tissue penetration, minimum cell damage, lack of interference from biological autofluorescence), and ratiometric imaging with a self-calibration feature to overcome systematic errors of intensity-only based fluorescent probes. In addition, the probes will possess good water solubility, high stability, excellent cell permeability, good biocompatibility, excellent intracellular retention, high selectivity, and sensitivity, as well as fast and reversible responses to pH changes. Seven undergraduate students will assist in conducting these experiments for each year of the proposal which will allow them to learn valuable synthetic and characterization research skills in this area. Undergraduate students will also conduct theoretical calculations, using the program Gaussian, to determine the absorptive properties of the probes.

抽象的 该项目采用计算指导方法，将出现比例计量荧光问题 在红色至近红外区域中运行，以准确确定细胞外和细胞内pH 细胞膜和不同细胞器的变化，例如溶酶体，线粒体，内质体 网状，高尔基体，并监测线粒体向溶酶体的递送 由养分饥饿，缺氧和药物治疗引起。问题的光谱化学属性将是 在合成之前确定，以判断所需光谱范围内的适用性。探针将包括 近红外荧光团连接到近红外犀牛染料，以实现对pH的比例响应 基于pH调制的变化导致问题的π结合变化。红色至近红外 荧光团将包括Bodipy染料，传统的近红外黄油染料及其衍生物，其中 黄黄核的中央氧原子将被其他元素（例如Si，s和N. sperolactone）代替 和可以调整为适当的PKA值与不同的PKA值的螺旋藻分子开关 研究的细胞器将被使用。针对不同细胞器和细胞膜的特定靶向将是 通过引入溶酶体特异性的吗啡，线粒体特异性的三苯基磷酸，高尔基 设备特异性苯甲酰胺，内质网特异性P-甲苯甲酰胺或细胞 膜特异性的两性骨zwitterion通过寡醇（乙二醇）垫片生存于荧光 基于螺旋藻开关的比率红色到近红外荧光探针将具有PKA 从4.5到6.2的值，仅在中性和基础下显示近红外荧光团的荧光 pH条件由于ronol部分在略微碱性条件下关闭了螺内酰胺环形结构 在线粒体内。溶酶体酸性环境将触发螺旋藻开口的开口 犀牛部分，显着增强了荧光团和杜鹃部分之间的π-结合 导致对pH变化的比率荧光响应。这些过程也将通过 进行合成之前的理论计算。这些问题将具有接近的总优势 红外成像（包括深层组织渗透，最小细胞损伤，生物学缺乏干扰 自动荧光）和具有自校准功能的比率成像，以克服系统错误 仅强度的荧光问题。此外，问题将具有良好的水溶性，高 稳定性，出色的细胞渗透性，良好的生物相容性，出色的细胞内保留率，高选择性， 和灵敏度，以及对pH变化的快速和可逆反应。七名本科生将协助 在为提案的每一年进行这些实验时，这将使他们能够学习有价值的合成 和表征该领域的研究技能。本科生还将进行理论 使用该程序高斯来确定问题的吸收性特性。