Local UV/VIS light generation for biomedical applications by NIR irradiation of targeted nanoparticles

通过目标纳米粒子的近红外辐射产生局部紫外/可见光，用于生物医学应用

• 批准号: 268984351
• 负责人: Professor Dr. Stefan H. Heinemann
• 金额: --
• 依托单位: Leibniz-Institut für Photonische Technologien (IPHT)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2015
• 资助国家: 德国
• 起止时间: 2014-12-31 至 2020-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/268984351?language=en
• 关键词: Local; UV; VIS; light; generation

Molecular signaling in cells requires a multitude of complex biochemical machineries relying on spatial and temporal separation as to allow the parallel processing of intracellular 'signals'. Studying such locally confined molecular signaling events thus affords their specific manipulation and detection. The proposed experimental study is conceived to achieve this goal by triggering photobiological reactions in the vicinity of identified proteins in the plasma membrane of living cells with high temporal resolution and narrow local confinement by use of nanometer scale UV/VIS light sources. The biological response will be monitored in a quantitative manner by the electrophysiological assessment of ion channel function employing patch-clamp technology. UV/VIS light (300-450 nm) is generated by optical frequency conversion of NIR laser light and spatially confined to the frequency converting nanoparticle, preferably up-converting nanocrystals (e.g. NaYF4:Yb,Tm under 980 nm diode laser irradiation) and, for comparison, BaTiO3 particles showing second harmonic generation under fs laser irradiation. Ultimately, the triggered photochemical reaction will be monitored by measuring the activity of individual ion channels with tailored properties. In a first set of applications, local uncaging of Ca2+ from photolabile chelators will be examined via Ca2+- and voltage-dependent K+ channel (Slo1 BK) activation and local production of reactive species by functional modulation of ROS-sensitive voltage-gated Na+ channels (roNaV), the latter being also used to assess the phototoxic burden. This ambitious goal will be approached in a stepwise manner by the combined action of closely co-operating physicists (optics and laser technology) and biophysicists/ electrophysiologists (biocompatibility assays and light impact on ion channels). The major challenges will be to optimize the yield of UV/VIS photons to allow for the reduction of the particle size from µm to nm, as well as to achieve molecular targeting of the nanoparticles to specific membrane proteins. Nanoparticle positioning will be assessed by high-resolution microscopy, while the impact of locally generated UV/VIS photons will be assessed indirectly via specific alteration of ion channel function, which is under tight electrophysiological control. This novel experimental approach avoids extensive hazardous UV irradiation of the entire cell. The proposed study will deepen our insight into membrane-delimited biochemical reactions and it will pave the way for new routine biomedical applications relying on photonic cell manipulation with molecular targeting precision. As a further spin-off, the non-linear optical properties of the frequency converting nanoparticles, not exhibiting photobleaching or blinking, may be used for superresolution microscopy (nanoscopy).

细胞中的分子信号传导需要依赖于空间和时间分离的大量复杂的生化机制，以允许细胞内“信号”的并行处理。因此，研究这种局部限制的分子信号传导事件提供了它们的特异性操纵和检测。拟议的实验研究被认为是实现这一目标的触发光生物学反应，在附近的确定蛋白质的活细胞质膜具有高的时间分辨率和窄的局部限制，通过使用纳米级的UV/维斯光源。将通过采用膜片钳技术对离子通道功能进行电生理学评估，以定量方式监测生物反应。UV/维斯光（300-450 nm）通过NIR激光的光学频率转换产生，并且在空间上被限制于频率转换纳米颗粒，优选上转换纳米晶体（例如在980 nm二极管激光照射下的NaYF 4：Yb，Tm），并且为了比较，BaTiO 3颗粒在fs激光照射下显示出二次谐波产生。最终，触发的光化学反应将通过测量具有定制特性的单个离子通道的活性来监测。在第一组应用中，将通过Ca 2+和电压依赖性K+通道（Slo 1 BK）激活以及通过对ROS敏感的电压门控Na+通道（roNaV）的功能调节来局部产生反应物质来检查Ca 2+从光不稳定螯合剂中的局部释放，后者也用于评估光毒性负担。这一雄心勃勃的目标将通过密切合作的物理学家（光学和激光技术）和生物物理学家/电生理学家（生物相容性测定和光对离子通道的影响）的联合行动逐步实现。主要的挑战将是优化UV/维斯光子的产率，以允许将颗粒尺寸从μm减小到nm，以及实现纳米颗粒对特定膜蛋白的分子靶向。纳米颗粒定位将通过高分辨率显微镜进行评估，而局部产生的UV/维斯光子的影响将通过离子通道功能的特异性改变进行间接评估，这是在严格的电生理控制下。这种新的实验方法避免了整个细胞的广泛的有害紫外线照射。这项研究将加深我们对膜界定的生化反应的了解，并将为依赖于具有分子靶向精度的光子细胞操作的新的常规生物医学应用铺平道路。作为进一步的副产品，频率转换纳米颗粒的非线性光学性质，不表现出光漂白或闪烁，可以用于超分辨率显微镜（纳米镜）。