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 G-protein-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: G-protein-coupled receptor; blood−brain barrier; nanosecond laser; plasmonic nanoparticle; protein inactivation.

Figure 1.

Schematic to illustrate the time and length scales of molecular hyperthermia (MH) compared with hyperthermia. Area left of the line represents thermal confinement. The thermal diffusion time is proportional to the square of size.

Figure 2.

Photo-inactivation of PAR2 by molecular hyperthermia. A) Schematic of PAR2 inactivation by molecular hyperthermia. B) Schematic illustrating the Ca²⁺ signaling due to PAR2 activation. G_αq is heterotrimeric G protein subunit, PLC is phospholipase C, IP₃ is inositol trisphosphate. C) Fluorescent immunocytochemistry imaging of HEK293 cells. Nucleus is in blue (Hoechst 33342), FRET Ca²⁺ sensor protein TN-XXL is in green, and PAR2-targeting antibody modified gold nanoparticle (AuNP-MAB3949) is in red (stained by Alexa 647 conjugated secondary antibody, indicated by white arrows), Control antibody modified gold nanoparticles (AuNP-Ctrl Ab) as a control show minimum signal (scale bar: 10 μm), AuNP concentration [AuNP] = 0.5 nM. D) The FRET ratio (ΔR/R) signal for different groups: (a) AuNP-Ctrl Ab, no laser; (b) AuNP-MAB3949, no laser; (c) AuNP-Ctrl Ab, with laser; (d) AuNP-MAB3949, with laser. [AuNP] = 0.32 nM. Laser conditions: 532 nm wavelength, fluence at 100 mJ/cm² and 10 pulses. The incubation time for AuNPs is 5 minutes. E) Dose response of PAR2 activity under different particle concentrations for different groups. The groups are color-coded as in Figure 2D. F) Dose-response curve for PAR2 activity under different laser fluence and pulse numbers. [AuNP] = 0.32 nM and AuNP incubation time is 5 minutes. G) PAR2 function recovery and cell proliferation after molecular hyperthermia treatment. The experimental conditions are the same as (d) group in Figure 2D. Laser were irradiated at 0 hour time point (indicated by red arrow). The normalized ΔR/R and cell proliferation indicate normalization with the control group (without AuNP incubation or laser treatment).

Figure 3.

High spatial resolution and molecular specificity for PAR2 photo-inactivation by molecular hyperthermia. A) Experimental design: after targeting PAR2 with AuNP-MAB3949, half of the cells were blocked from laser irradiation. B) Experimental setup including 2-photon imaging of cytoplasmic Ca²⁺ release. C) Fluorescence images of cells after laser irradiation (scale bar: 50 μm). The line indicates the border of laser irradiation, with the bottom left side receiving laser irradiation (ROI in red) and the top right side without laser irradiation (ROI in blue). D) Ca²⁺ signals for different ROIs. The red line is the FRET ratio from the cells in the red semicircle (with laser) and the blue line indicates the FRET ratio from the blue semicircle (no laser). ACSF refers to artificial cerebral spinal fluid. E) Schematic to illustrate the photo-inactivation of PAR2 without compromising somatostatin receptor 2 (SSTR2) activity. F) PAR2 and SSTR2 activity after molecular hyperthermia treatment. [AuNP] = 0.32 nM and AuNP incubation time is 5 minutes. Laser conditions: 532 nm wavelength, fluence at 100 mJ/cm² and 10 pulses.

Figure 4.

Photo-inactivation of a tight junction protein (JAM-A) by molecular hyperthermia to manipulate the blood-brain barrier (BBB). A) Schematic illustrating the JAM-A photo-inactivation by molecular hyperthermia. B) Fluorescent immunocytochemistry staining for brain endothelial cells (hCMEC/D3) targeted by AuNP-BV16. Nucleus is in blue and JAM-A is in green (Alexa 488) (scale bar: 20 μm). C) Cell proliferation measurements with and without laser irradiation and AuNP incubation. Ctrl indicates control group (no AuNP). D) Schematic illustrating the transendothelial electrical resistance (TEER) measurement. E) TEER measurements before and after molecular hyperthermia. F) Schematic of permeability measurement. G) Permeability measurements of different sized macromolecules diffusing cross the cellular barrier. FD-4 indicates 4 kDa FITC-labeled dextran.

Figure 5.

Protein inactivation by molecular hyperthermia (MH) does not lead to nanobubble generation. (A) Protein inactivation and nanobubble generation probability as a function of laser fluence. Photo-inactivation of α-chymotrypsin (α-Cht) by 30 nm AuNP (in solution) is adapted with permission from ref . Copyright 2017 John Wiley and Sons. The blue dash and solid lines are Boltzmann fitting for nanobubble probability measurements for 30 and 45 nm AuNP respectively. The blue shaded zone indicates >5% bubble generation probability. (B) Laser fluence at 50% protein inactivation and nanobubble probability.