Optical control of neuronal activities with photoswitchable nanovesicles

Abstract

Precise modulation of neuronal activity by neuroactive molecules is essential for understanding brain circuits and behavior. However, tools for highly controllable molecular release are lacking. Here, we developed a photoswitchable nanovesicle with azobenzene-containing phosphatidylcholine (azo-PC), coined 'azosome', for neuromodulation. Irradiation with 365 nm light triggers the trans-to-cis isomerization of azo-PC, resulting in a disordered lipid bilayer with decreased thickness and cargo release. Irradiation with 455 nm light induces reverse isomerization and switches the release off. Real-time fluorescence imaging shows controllable and repeatable cargo release within seconds (< 3 s). Importantly, we demonstrate that SKF-81297, a dopamine D1-receptor agonist, can be repeatedly released from the azosome to activate cultures of primary striatal neurons. Azosome shows promise for precise optical control over the molecular release and can be a valuable tool for molecular neuroscience studies.

Keywords: azobenzene; controlled release; liposome; neuromodulation; photoswitch.

Figure 1

Photophysical properties of asozomes. (a) Schematic of the photoisomerization of azo-PC. (b) Cryo-TEM image of azosomes. Scale bar: 100 nm. (c) and (d) UV–Vis spectra change of azosome upon the irradiation of (c) 365 nm light and (d) 455 nm light. (e) The absorbance change of azosome at 326 and 455 nm light as a function of irradiation time. (f) and (g) UV–Vis spectra change of azosome in the dark at (f) 22 °C and (g) 37 °C. 365 nm light irradiation (25 mW/cm², 60 s) was performed on the azosome to switch trans-azo-PC to cis-azo-PC at 0 min. (h) The absorbance change of azosome at 326 nm over time in the dark at 22, 37 and 45 °C. The data were fit based on a first-order reaction to give the half-life of cis-azo-PC. (i) The Z-average hydrodynamic diameter of azosome (azo-PC: 25%) measured by dynamic light scattering (DLS) under photoisomerization of azo-PC. 365 or 455 nm light was irradiated on the azosome for 30 s at 25 mW/cm².

Figure 2

Molecular dynamics simulation for azo-PC lipid bilayer. (a) Snapshots of the trans and cis bilayers. Selected azobenzene groups are highlighted in light cyan with the ring planes in red. Lipid tails are shown in transparent gray. The blue and brown spheres represent nitrogen and phosphorous atoms on the head group, respectively. Water is represented as light purple regions. (b) The lipid atom density profile of the azo-PC bilayer as a function of the distance from the bilayer center. The orange and blue lines represent the trans-azo-PC and cis-azo-PC bilayers. Solid lines: the overall lipid density; dashed lines: lipid head groups; shaded regions: azobenzene groups. (c) Area per lipid in the tensionless ensemble of the azo-PC bilayer when the isomeric state of every lipid is altered between cis and trans every 100 ns. (d) Experimental and simulated azosome area increase with different percentages of azo-PC after the irradiation with 365 nm light (25 mW/cm², 30 s). Data are expressed as mean ± standard deviation (S.D.) Simulation data were extrapolated to 0% azo-PC from the data in (c).

Figure 3

Photoswitchable fluorophore release from asozomes. (a) Schematic of the photoswitchable release from azosome. (b) Plot of calcein release efficiency of azosomes with different percentages of azo-PC upon 365 nm light irradiation (10 mW/cm²). (c) Plot of calcein release efficiency from azosomes at different 365 nm light intensities and durations. Calcein fluorescence was measured 30 s after irradiation in (b) and (c). (d) Real-time fluorescence intensity of calcein (ΔF) plotted over time under the sequential irradiations of 365 and 455 nm light (40 mW/cm²). The purple and blue rectangles indicate 365 and 455 nm light irradiation, respectively. (e) Real-time fluorescence intensity of calcein (ΔF) plotted over time under multiple cycles of irradiations. The purple and blue arrows indicate the sequential irradiations of 365 and 455 nm light every 5 s (40 mW/cm²). Data are expressed as mean ± S.D.

Figure 4

Controlled release of a D1 receptor agonist for neuromodulation. (a) Schematic photorelease of SKF-81297 from the azosome to induce Ca²⁺ elevation in primary mouse striatal neurons. (b) Real-time fluorescent images of primary mouse striatal neurons before and after the irradiation of sequential 365 nm light and 455 nm light. Fluo-4 was used as the Ca²⁺ indicator. Scale bar: 50 μm. (c) and (d) The fluorescence change (ΔF/F) plotted as a function of time from individual neurons incubated with (c) SKF-azosome or (d) SKF-liposome. The irradiation of 365 and 455 nm light was performed during the imaging at 0 s. Three repeated irradiations (25 mW/cm²) were performed on SKF-azosome with an interval of 10 min (1^st, 365 nm 0.3 s + 455 nm 0.3 s; 2^nd, 365 nm 0.5 s + 455 nm 0.5 s; 3^rd, 365 nm 1 s + 455 nm 1 s). The irradiation condition (25 mW/cm²) in (d) was 365 nm 1 s + 455 nm 1 s. The activated neuron was counted if the mean fluorescence change (ΔF/F) after stimulation was larger than the mean + 3δ (δ: standard deviation) of baseline. The neurons were labeled in the same order in (c).s