Plasmonic Stimulation of Gold Nanorods for the Photothermal Control of Engineered Living Materials

Composites of living cells in polymer gel matrices are called “Living Materials”[1]. They are useful for the controlled release of drugs that are produced by the cells while protecting them and their environment. Here, we demonstrate an optically triggered Living Material based on thermoresponsive bacteria. A “core” of a pluronic hydrogel with engineered <i>Escherichia coli</i> was enclosed in “shell” of a composite hydrogel that contained gold nanorods (AuNR) that were 41 nm long and 10 nm in diameter. The absorption spectrum of the AuNR coincides with the transparent window of human tissues. The plasmonic rods absorb laser light, heat the shell, and activate the bacteria, inducing the production of mCherry proteins or darobactin. Living materials were prepared in structured microslides that enabled both quantitative studies of spatial and temporal temperature variation and protein expression.<br/>First, we characterized the thermoplasmonic composite and determined its overall photothermal conversion efficiency and the resulting thermal response with a AuNR concentration equivalent to an optical density of OD_{808 nm} = 4. Infrared thermography and embedded thermocouples were combined to obtain temperature distributions at a sampling rate of 0.2 Hz and an accuracy of 0.1 K. Illumination with a collimated laser spot of 5 mm diameter at 808 nm heated the cylindrical core-shell Living Material from an initial 20°C to a steady-state temperature of 40°C in air in 300 seconds. The IR camera indicated a lateral temperature gradient from center to the border with a maximum temperature difference of 4.7°C.<br/>The genetically engineered <i>E. coli</i> contained in the core were activated by heating and produced the fluorescent protein mCherry. In a second set of experiment, we quantified its production as a function of laser power, time, and position via <i>ex situ</i> fluorescence microscope. The fluorescence intensity correlated with the temperature distribution measured above. The maximal protein expression rate for the living material with this <i>E. Coli</i> strain was found to be between 39-44°C, consistent with reference experiments in suspensions. Protein production at 0.6 W/m² was maximal at the center of the illuminated area and declined at its borders, where temperature dropped. Increasing laser power first led to an increase of fluorescence intensity at the borders. A uniform fluorescence intensity was reached across the illuminated area at 0.7 W/m². Protein expression continued for at least 4 h, with a linear increase of fluorescence that indicated sustained expression activity of the bacteria. Further increasing power density reduced the fluorescence intensity at the center. The decline occurred when the center temperature exceeded 45°C, the maximum viable temperature for the bacteria.<br/>In a third set of experiments, we compared the results obtained for the model compound mCherry to the antibiotic darobactin that was produced by a different strain of engineered <i>E. Coli</i>. After photothermal triggering, darobactin was expressed, diffused through the gel, and into surrounding media, where we detected its presence via ESI-MS as a function of laser irradiation time. The results will be discussed in view of possible applications in therapies for chronical inflammations, e.g. in periodontitis.<br/> <br/>Acknowledgement:<br/>We thank Shardul Bhusari for providing the hydrogel.<br/> <br/>References:<br/>[1] Nguyen, P. Q., N. D. Courchesne, A. Duraj-Thatte, P. Praveschotinunt & N. S. Joshi, Adv. Mater., 2018, 30, e1704847.