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

• Applicants: Professor Dr. Stefan H. Heinemann; Professor Dr. Rainer Heintzmann; Professor Dr. Herbert Stafast
• Subject Area: Biophysics
• Term: from 2015 to 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 268984351

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).

• DFG Programme: Research Grants