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

细胞中的分子信号需要大量复杂的生化机制，这些机制依赖于空间和时间的分离，以允许细胞内“信号”的并行处理。因此，研究这种局部受限的分子信号事件提供了它们的特定操作和检测。该实验研究旨在通过使用纳米级UV/VIS光源，在高时间分辨率和狭窄的局部限制下，触发活细胞质膜中鉴定蛋白附近的光生物反应，从而实现这一目标。生物反应将通过采用膜片钳技术对离子通道功能进行电生理评估，以定量的方式进行监测。UV/VIS光（300-450 nm）是由近红外激光的光学频率转换产生的，并且在空间上局限于频率转换的纳米粒子，最好是上转换的纳米晶体（例如在980 nm二极管激光照射下的NaYF4:Yb，Tm），相比之下，BaTiO3粒子在fs激光照射下表现出二次谐波产生。最终，将通过测量具有特定性质的单个离子通道的活性来监测触发的光化学反应。在第一组应用中，将通过Ca2+和电压依赖的K+通道（Slo1 BK）激活和ros敏感电压门控Na+通道（roNaV）的功能调节来检查光致螯合剂对Ca2+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紧密合作的物理学家（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/VIS光子的产量，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。纳米粒子定位将通过高分辨率显微镜进行评估，而局部产生的UV/VIS光子的影响将通过离子通道功能的特定改变间接评估，这是在严格的电生理控制下进行的。这种新颖的实验方法避免了整个细胞广泛的有害紫外线照射。该研究将加深我们对膜界生物化学反应的认识，并将为基于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。作为进一步的衍生产物，频率转换纳米粒子的非线性光学特性，不表现出光漂白或闪烁，可以用于超分辨率显微镜（纳米显微镜）。

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).