Modulation of Local Cellular Activities using a Photothermal Dye-Based Subcellular-Sized Heat Spot

Abstract

Thermal engineering at the microscale, such as the regulation and precise evaluation of the temperature within cellular environments, is a major challenge for basic biological research and biomaterials development. We engineered a polymeric nanoparticle having a fluorescent temperature sensory dye and a photothermal dye embedded in the polymer matrix, named nanoheater-thermometer (nanoHT). When nanoHT is illuminated with a near-infrared laser at 808 nm, a subcellular-sized heat spot is generated in a live cell. Fluorescence thermometry allows the temperature increment to be read out concurrently at individual heat spots. Within a few seconds of an increase in temperature by approximately 11.4 °C from the base temperature (37 °C), we observed the death of HeLa cells. The cell death was observed to be triggered from the exact local heat spot at the subcellular level under the fluorescence microscope. Furthermore, we demonstrate the application of nanoHT for the induction of muscle contraction in C2C12 myotubes by heat release. We successfully showed heat-induced contraction to occur in a limited area of a single myotube based on the alteration of protein-protein interactions related to the contraction event. These results demonstrate that even a single heat spot provided by a photothermal material can be extremely effective in altering cellular functions.

Keywords: NIR light; cell engineering; local heating; nanoheater-thermometer; photothermal dye.

Figure 1

Characterization of nanoHT with regard to the ability of heat release and temperature sensing. (A) Schematic illustrations of nanoHT and its controlled heating inside a cell. (B) Excitation and fluorescence spectra of C102 and EuDT, and the absorption spectrum of V-Nc in nanoHT. (C) DLS measurement of nanoHT. The average of diameter: 153 ± 51 nm (mean ± SD). The black line indicates the log-normal fitting curve. (D) The normalized fluorescence intensity (FI) values of C102 and EuDT were plotted against temperature as the first axis, and the ratio value (EuDT/C102), which is normalized to that of 37 °C, as the second axis. Error bars, SD (n = 3). The temperature sensitivities of C102, EuDT, and the ratio obtained from the slopes were determined to −0.06, −2.96, and −2.89%/°C, respectively. (E) The evaluation of the heating ability of nanoHT suspension in the cuvette by irradiation with an NIR laser (808 nm). Error bars, SD (n = 3).

Figure 2

Validation of heat-releasing ability of nanoHT. (A) Schematic representation of the setup to validate the temperature-sensing ability of nanoHT under the microscope with an NIR infrared laser (980 nm). Scale bar: 5 μm. (B) The mean fluorescence intensities of C102 and EuDT at each ROI as shown in (A) were plotted every 0.56 s in the time course (5 s NIR laser stimulation). (C) The calibration curve of nanoHT against temperature obtained under the microscopic observation. Error bars, SD (n = 3). (D) The normalized ratio (EuDT/C102) was converted to the temperature increment (ΔT) profile using the calibration curve. (E, F) Validation of the heat-releasing ability of nanoHT using an 808 nm laser. The mean fluorescence intensity at each ROI was plotted in the time course with NIR laser stimulation being performed for 5 s intervals at different laser powers (0.98–11 mW). (G) The average of temperature increment provided by nanoHT (error bars, SD n = 10) was plotted for each value of laser power of the 808 nm NIR laser. Solid line shows the linear fit. White dotted circles in (A) and (E) indicate the NIR spots.

Figure 3

Validation of heat-releasing capabilities of nanoHT in HeLa cells. (A, B) Validation of the heat-releasing ability of nanoHT using an 808 nm laser. The mean fluorescence intensity of nanoHT in (A) was plotted in the time course with NIR laser stimulation being performed for 5 s intervals at different laser powers (0.98–11 mW). Scale bar: 10 μm. (C) The averages of temperature increment provided by nanoHT (error bars, SD n = 12) were plotted at varying laser powers of the 808 nm NIR laser. Solid line shows the linear fit. (D) The different temporal patterns of the temperature increment created by nanoHT. The mean of the normalized ratio of nanoHT with SD (n = 3) was plotted in the time course. (E) Colocalization test with a lysosome tracker in the upper panel (red: C102, green: lysosome tracker to stain acidic organelles). Enlarged view of the region surrounded by a dashed square before, during, and after heating. Scale bar: 10 μm. White dotted circles in (A) and (E) indicate the NIR spots.

Figure 4

Evaluation of temperature distribution provided by nanoHT in a HeLa cell and in the dish. (A) nanoHT was located at the surface of the dish filled with the blue fluorescent protein (BFP) solution, while nanoHT was taken into the HeLa cell expressing BFP. The trajectory of nanoHT is depicted in the lower panel in the dish (left side) and HeLa cell (right side), respectively. During the 50 s tracking, the NIR laser stimulation was performed at three different powers (2.2, 6.6, and 11.2 mW) for 5 s intervals. (B) The total traveling distance of nanoHT in the dish and HeLa cell during 50 s. The data set corresponds to Figure 4A. The linear fitting curves were y = 0.03x + 0.13 (R² = 0.87, 2.2 mW), y = 0.03x + 0.13 (R² = 0.98, 6.6 mW), and y = 0.03x + 0.13 (R² = 0.98, 11.2 mW) in the dish; y = 0.09x – 0.08 (R² = 0.95, 2.2 mW), y = 0.13x – 0.72 (R² = 0.95, 6.6 mW), and y = 0.12x – 0.64 (R² = 0.95, 11.2 mW) in HeLa cell. (C) The velocity of nanoHT (μm/s) during heating is plotted at different temperatures in the dish and HeLa cell. ΔT represents mean ± SD for 5 s heating. (D) The analysis of temperature distribution generated by nanoHT using BFP at different laser powers (2.2, 6.6, and 11.2 mW). The grouped stacked images during 5 s heating were divided by the image before heating. The triangle marks indicated the position of the line profile as shown at the bottom of each image.

Figure 5

Heat-triggered cell death by nanoHT. (A) Dual imaging of Apopxin Green (apoptosis marker) and nanoHT (blue: C102) in a HeLa cell. (B) The time course of the normalized ratio of nanoHT and fluorescence of Apopxin Green in the vicinity of the heat spot (NIR stimulation for 10 s). The temperature increments were estimated by the calibration curve. (C) Images of the HeLa cell stained with Apopxin Green and PI (for detection of necrosis or late stage of apoptosis) after heating (1 and 10 min). (D) The correlation between the temperature increment of nanoHT and the enhancement of apopxin green (F/F₀). Laser power was varied from 8.8 to 11.2 mW. ΔT represents mean ± SD for 10 s of heating. (E) Dual imaging with Ca²⁺ (B-GECO) and Apopxin Green in a HeLa cell. Elapsed time is shown in the top left of each image in (A) and (E). Scale bars: 10 μm. White dotted circles in (A), (C), and (E) indicate the NIR spots.

Figure 6

Evaluation of intracellular ATP dynamics during local heating. (A) Morphological changes occur in mitochondria after heating at the temperature above the threshold temperature to induce the cell death. Scale bar: 20 μm. (B) Fluorescence image of HeLa cell expressing MaLionG (cytoplasmic ATP) and mitoMaLionR (mitochondrial ATP) with nanoHT (C102). Scale bar: 20 μm. (C) The time course of mitoMaLionR in the vicinity of nanoHT (the local area of the cell shown in (B)). Scale bar: 10 μm. (D) The ATP dynamics in cytoplasm (MaLionG) and mitochondria (mitoMaLionR) at ROI1 and -2 of the same cell shown in (B). The heating period is 1 min. (E) The analysis of mitochondrial ATP dynamics in the vicinity of the heat spot similar to ROI1 shown in (B) in four cells at different temperature increments (3.6 ± 1.5, 5.3 ± 0.5, 8.7 ± 0.3, and 10.1 ± 0.7 °C below the threshold of the cell death). (F) The thick lines of MaLionG and mitoMaLionR represent the average of seven cells with SD at different temperatures. White dotted circles in (A–C) indicate the NIR spots.

Figure 7

C2C12 myotube contraction induced by sequential heating by nanoHT. (A) Images of C2C12 myotube with nanoHT (red) and CellTracker Green (green: cytoplasm). Scale bar: 10 μm. (B) Temperature increments provided by sequential NIR stimulation every 5 s. (C) Kymographs of line A and B as shown in (A). (D) The dynamic profile of the line A in response to NIR stimulation. (E) Quantitative analysis of the displacement induced by the heating by nanoHT. Left: schematic illustrations showing the image analysis of the morphology change of the C2C12 myotube by heating. Center: the x–z profile of line A. Each dot shows the average with SD during 5 s. Right: the maximum displacement at the x-axis was plotted against varying temperature. The solid line represents the exponential fit. ΔT represents mean ± SD for 5 s of heating. A white dotted circle indicates the NIR spot.