Transient Photoinactivation of Cell Membrane Protein Activity without Genetic Modification by Molecular Hyperthermia

Abstract

Precise manipulation of protein activity in living systems has broad applications in biomedical sciences. However, it is challenging to use light to manipulate protein activity in living systems without genetic modification. Here, we report a technique to optically switch off protein activity in living cells with high spatiotemporal resolution, referred to as molecular hyperthermia (MH). MH is based on the nanoscale-confined heating of plasmonic gold nanoparticles by short laser pulses to unfold and photoinactivate targeted proteins of interest. First, we show that protease-activated receptor 2 (PAR2), a Gprotein-coupled 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 laser-targeted cells without affecting surrounding cells. Furthermore, we demonstrate the molecular specificity of MH by inactivating PAR2 while leaving other receptors intact. Second, we demonstrate 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. MH is distinct from traditional hyperthermia (that induces global tissue heating) in both its time and length scales: nanoseconds versus seconds, nanometers versus millimeters. Our results demonstrate that MH enables selective and remote manipulation of protein activity and cellular behavior without genetic modification. KEYWORDS: plasmonic nanoparticle, nanosecond laser, protein inactivation, G-protein-coupled receptor, blood−brain barrier S elective and remote manipulation of protein function in living systems is important to elucidate the protein function and develop precise therapeutics for disease treatment. Advances in tool development have made

Computational Investigation of Protein Photoinactivation by Molecular Hyperthermia

“…MH selectively inactivates enzymes targeted by AuNS and leaves untargeted enzymes intact, demonstrating the spatial selectivity of MH [25,27]. We further showed that MH can be used to inactivate cell membrane proteins in live cells without compromising cell viability [26]. MH is a promising method to selectively and remotely manipulate protein activity and cellular behavior.…”

“…GPCRs is a large protein family and contains many potential drug targeting sites for disease treatments. In our previous study, we have investigated protease activated receptor 2 (PAR2), which is also a GPCR [26]. Although kinetic parameters vary from protein to protein, impact zones of opsin and related receptors suggest MH can be a useful method to inactivate GPCRs.…”

“…Recently, we developed a method called molecular hyperthermia (MH) to photoinactivate protein activity [25,26] by taking advantage of nanoscale plasmonic heating. MH utilizes nanosecond laser pulses to excite plasmonic nanoparticle, for example gold nanosphere (AuNS), as a nanoheater to thermally inactivate proteins adjacent to the nanoparticle.…”

Single Pulse Heating of Nanoparticle Array for Biological Applications

“…Heating generated by nanomaterials broadly falls into one of two regimes: nanoscale-confined heating (NCH) and macroscale-collective heating (MCH). NCH is characterized by local temperature increases at the nanoscale which remain confined near the surface of the nanoparticle, as in the case of controlling protein activity by molecular hyperthermia, [15,16,26] or membrane permeability by opto-poration. [16,[26][27][28] In contrast, MCH is characterized by a widespread temperature increase throughout the medium, such as whole cells or tissues.…”

Nanoparticle fragmentation at solid state under single picosecond laser pulse stimulation

“…These phenomena have been harnessed for a variety of applications. For example, near-field enhancement can be used for optical trapping of molecules 4 , while surface-enhanced Raman scattering and photothermal effects have found applications in imaging 5, 6 , sensing 7, 8, 9, 10 and actuation of biological activities 11, 12 . However, nanoparticles subjected to pulsed laser irradiation may also undergo laser-induced fragmentation, in which the original nanoparticles are fragmented into smaller particles 13 .…”