Backstage of rising body temperature: Advances in research on intracellular heat diffusion

Comment on: Sotoma, S., Zhong, C., Kah, J.C.Y., Yamashita, H., Plakhotnik, T., Harada, Y., Suzuki, M. In situ measurements of intracellular thermal conductivity using heater-thermometer hybrid diamond nanosensors. Sci Adv. 2021;7(3): eabd7888

Intracellular process is a fundamental origin of rising body temperature above the ambient value. However, thermal effects in an isolated cell are very difficult to investigate. A reason for this difficulty is a simple fact that heat escapes from a hot body through its surface but it is generated in the body volume. Because the volume grows faster than the surface with increasing the size of an object (for example, the surface of a sphere increases four times if the radius of the sphere doubles while the corresponding increase of the volume is eight-fold), the expected thermal effects in a microscopic cell are orders of magnitude smaller than the temperature rise in a large organism composed of many cells. This difficulty has been eased recently with development of thermal nanosensors. Since then, several groups have reported very surprising results indicating that the temperature elevation inside cells above the ambient conditions was at least a thousand times larger than expected (see review by Suzuki & Plakhotnik [1]). A recent publication by Sotoma et al. [2] demonstrates a new advance in the methodology where a single nanoprobe is equipped with a thermometer and a heater (Figure 1). Such a dual-purpose composite nanoparticle can be used to study heat diffusion inside cells and has been applied to study two cell lines, HeLa and MCF-7. A stunning discovery of this work was that the thermal conductivity in these cells is significantly hindered (a factor of 5–6 times smaller) in comparison to thermal conductivity in water or a buffer solution. A microscopic theory explaining such a reduction is not yet available. Fundamental understanding of the intracellular heat transfer will require advance modeling of molecular dynamics (which is a big challenge for modern computers to do on a scale comparable to the cell size) and more control on positioning of the nanoprobe inside cells (at present the probe particles are internalized by endocytosis and their localization is not controlled but is determined at random).

Figure 1. Temperature at different scales. Transmission electron microscopy image in the center of panel a shows a PDA-FND nanoprobe of about a 200-nm in diameter used in our study [2] and designed to measure temperature and thermal conductivity in a spherical shell on the order of 100 nm thick surrounding the particle. The principles of operation for PDA-FND nanoprobes are outlined in the text. Panel b shows a fluorescence image of a HeLa cell (image size 42 × 42 μm; plasma membrane is colored in green) with a PDA-FND probe (the magenta dot in the corner made of the dashed lines). On the scale from one cell to a tissue and to an entire organism (b, c, and d), the magnitude of the plausible temperature rise increases with the size of the object, but the difficulty of measurements increases as the size shrinks

The hybrid probe presented in [2] is made of a diamond nanocrystal of about 100 nm in diameter enclosed in an approximately 50 nm thick polymer shell. When the particle is illuminated with green excitation light, the polymer (polydopamine or PDA) absorbs a fraction of such light and converts it into heat which is released into the media surrounding the particle, while the diamond emits light at a different color (dark red) and responds to a change of temperature. Due to such emission, the diamonds are called fluorescent nanodiamonds (FNDs). The dark-red light can be detected even if emitted by a single nanoparticle.

The measurements are done by a method called optically detected magnetic resonance (ODMR). The light intensity serves as an indicator of a resonance between a paramagnetic absorption line of an impurity embedded in diamond crystal (called nitrogen-vacancy center because it consists of a nitrogen atom replacing on atom of carbon and a vacancy in the crystal lattice) and microwave radiation. Luminescence intensity decreases when the microwave frequency is set to a specific (resonance) value, but such resonance frequency depends on the temperature. Thus, one can infer the temperature of the diamond crystal (which depends on the cooling rate of the crystal) optically by measuring light intensity emitted by an FND at different frequencies of the microwave radiation. The cooling rate depends on the heat transfer process in the particle environment and thus the value of the thermal conductivity can be obtained.

About 60 cells (30 from each of the two cell lines) have been tested with PDA-FND nanoprobes in the experiment. Each cell has contained an absolute minimum of probe particles required for the measurements with a minimal disturbance. The averaged values of the thermal conductivity obtained using two different batches of PDA-FND nanoprobes in both lines of cells and in various control media (air, water and oil) were similar and confirmed batch-to-batch reproducibility of the values determined. However, more work needs to be done to reduce the distribution of particle sizes within one batch and thus to exclude the time-consuming averaging from the measurement protocol.

Further research on intracellular processes using microscopic tools such as PDA-FND nanoprobes may extend the role of cellular thermogenesis in biology beyond its conventionally assumed contribution to rising tissue and body temperature. A comprehensive review by Kiyatkin [3] describes how the brain hyperthermia caused by external stimuli and by neural activity affects brain structure and functions. Endogenous thermogenesis in other specialized tissues such as skeletal muscle is also physiologically important. Tools like PDA-FND may help to reveal how the endogenous heat controls processes at the subcellular scale. Although it is generally accepted that processes in cells are regulated by signaling cascades of biochemical reactions and diffusion of molecules, we believe that thermal signaling could also be an important channel for this purpose.

Knowledge of intracellular heat dynamics and the extent to which the cellular architecture may affect the heat flow will allow us to draw an accurate and complex picture of intracellular heat diffusion. Although the body temperature is routinely measured by medical practitioners, a sophisticated approach is needed for minimally invasive monitoring of internal body temperature in applications such as hyperthermia treatment [4,5]. Such control helps to confirm the achieved temperature at the targeted site and to monitor the unwanted temperature rise in non-targeted regions during the treatment. Sensors similar to the one described in this paper but adapted for clinical use will have much higher spatial resolution than provided by near-infrared imaging and spectroscopy explored in [4,5] and may be a useful addition to the treatment protocol. Measurements of thermal conductivity demonstrated in [2] help predict transient temperature rises at the cellular sites of thermogenesis, the magnitude of arising thermal gradients and the subsequent thermally induced processes in their neighborhood [1]. Zaretsky et al. [6] investigated temperature of the arterial blood flow and of the surrounding tissue. Their mathematical model based on macroscopic equation of heat diffusion predicts thermal gradients of up to 1.3°C. In principle, the intracellular thermal conductivity that we have investigated [1,2] can be related to the macroscopic values at the tissue scale, the essential parameters of the model. Ultimately, all local (cellular scale) thermal phenomena can be linked to the processes on the scale of tissue and organismal to form a united picture (Figure 1).