Photoinactivation of Proteins by Molecular Hyperthermia
Spatiotemporal control of protein structure and activity in biological systems has important and broad implications in biomedical sciences as evidenced by recent advances in optogenetic approaches. However, it is challenging to use light to manipulate protein activity in living systems without genetic modification. Plasmonic nanoparticles, due to their unique optical properties, provide exceptional nano-bio interface to control biological activities. Upon laser irradiation, plasmonic nanoparticles converse light energy into heat. This unique energy conversion pathway, known as plasmonic heating, can be precisely engineered on different scales by tuning the laser properties such as pulse duration and energy. Theoretically, it is possible to generate a nanoscale plasmonic heating by using the short laser pulse. In this work, we demonstrated that the nanoscale plasmonic heating can be used to precisely photoinactivate protein activities. Firstly, we demonstrated that nanosecond pulsed laser heating of gold nanoparticles (AuNP) leads to an ultrahigh and ultrashort temperature increase, coined as “molecular hyperthermia” (MH). MH causes selective unfolding and inactivation of proteins adjacent to the AuNP. Protein inactivation is highly dependent on both laser pulse energy and AuNP size, and has a well-defined impact zone in the nanometer scale. We observed aggregation behavior of protein–AuNP conjugates at high laser intensities that originates from significant protein unfolding. Secondly, we demonstrated MH can optically switch off protein activity in living cells with high spatiotemporal resolution. We showed that protease-activated receptor 2 (PAR2), a G-proteincoupled receptor and an important pathway that leads to pain sensitization, can be photoinactivated in situ by MH without compromising cell proliferation. PAR2 activity can be switched off in lasertargeted cells without affecting surrounding cells. Furthermore, we demonstrated the molecular specificity of MH by inactivating PAR2 while leaving other receptors intact. Next, we demonstrated that the photoinactivation of a tight junction protein in brain endothelial monolayers leads to a reversible blood–brain barrier opening in vitro. Lastly, the protein inactivation by MH is below the nanobubble generation threshold and thus is predominantly due to the nanoscale heating. Thirdly, we used a numerical model to study important parameters and conditions for MH to efficiently inactivate proteins in the nanoscale. To quantify the protein inactivation process, impact zone is defined as the range where proteins will be inactivated by nanoparticle localized heating. We found that stretching the laser pulse duration reduces the MH impact zone with the same laser pulse energy. Temperature-dependent material density and specific heat have little effect on MH, while temperature-dependent thermal conductivity decreases the impact zone compared with constant properties. The thermal interface resistance doesn’t have effect on MH water below 10-8 K m2 W-1. Lastly, study of nanoparticle geometry suggests that nanosphere has larger impact volume than nanorods with the same particle volume and energy input. In summary, MH enables selective and remote manipulation of protein activity and cellular behavior. MH is a promising method with broad applicability to switch off protein activity without genetic modification and will find many applications in biomedical sciences.