To measure the light-to-heat conversion, we prepared two different kinds of samples: one artificial brain phantom and one from dissected pig brain (Fig. 1a). The samples were irradiated with a NIR laser with a wavelength of 806 nm and the temperature (Fig. 1b) was recorded by an infrared (thermal) camera. Fig. 1c is an example of such a thermal image, where the temperature, T, is defined as the maximum temperature within the region of interest (white square).
Plasmonic photothermal heating of artificial tissue
We experimentally determined the heating of different concentrations of AuNSs in artificial tissue, i.e., a 0.6% agarose solution (Eldridge et al. 2016), and a control without AuNSs. Fig. 2a shows temperature change, \(\Delta T\), versus time, t, of the phantom tissue for varying particle concentration. The tissue was heated with a 1.5 W laser for half an hour and cooled (laser off) for another half an hour. The temperature driving force approximation (Bardhan et al. 2009; Roper et al. 2007; Pattani and Tunnel 2013) permits the system equilibrium time, \(\tau\), and the steady-state temperature, \(T_\text {ss}\), to be estimated using system parameters from heating data and cooling data, respectively. For the heating, we found the following exponential dependence on t (Roper et al. 2007):
$$\begin{aligned} \Delta T=\Delta T_\text {ss}(1-e^{-t/\tau }) \end{aligned}$$
(1)
and for cooling:
$$\begin{aligned} \Delta T=\Delta T_\text {ss}e^{-t/\tau }. \end{aligned}$$
(2)
It is worth noticing that Eqs. (1) and (2) both are independent of the ambient temperatures. Thus, the results from this study, which were all obtained at room temperature, are also applicable at body temperature (\(37\,^{\circ }\hbox {C}\)). For the phantom tissue without AuNSs, we found \(\Delta T_\text {ss} = 1.7\hbox { K}\) (Fig. 2a). For a tissue at an ambient temperature of \(37\,^{\circ }\hbox {C}\), this corresponds to moderate hyperthermia. In contrast, we measure extreme hyperthermia (\(\Delta T> 5^{\circ }\) K) with 5.6 \(\upmu\)g/ml AuNS and a laser power of 1.5 W: \(\Delta T_\text {ss}=7.8\) K.
In a similar experiment (Fig. 2b), we kept the nanoparticle density constant at 5.6 \(\upmu\)g/ml and varied the laser power in the range from 0.5 to 2 W. Heating rates can be deducted from the slopes of Fig. 2b and were found to be (\(6.9\pm 4.2\)) K/W for AgNP and (\(4.7\pm 1.3\)) K/W for AuNSs. Hence, in artificial tissue, the AgNPs are the most efficient nano-heaters but both nanoparticle suspension heats significantly more than the control. There is a pronounced difference in the temperature achieved and, hence, of the effect of the PTTP, depending on whether the phantom is a suspension of plasmonic nanoparticles or not. It is worth noticing that for all investigated concentrations, 50% of \(\Delta T_\text {ss}\) is reached within the first 7 min of irradiation. Hence, hyperthermia is reached soon after the laser is turned on.
NIR laser-induced heating of cerebrum, cerebellum, and brain stem tissue
Paralleling the experiments in phantom tissue, we investigated the temperature increase of three different types of porcine brain tissue under laser irradiation. Due to the in-homogeneous distribution of gray matter and white matter in the different samples (Fig. 1b) and in the cerebrum samples in particular, we consistently irradiated the samples from the same side to reduce the variance (cerebrum samples were irradiated from the cortex side). An example of the temperature evolution is given in Fig. 3a. The thermal reaction of the brain tissue is represented by the steady-state temperature \(\Delta T_\text {ss}\), which was found by fitting the heating curve of the irradiated sample with Eqs. (1) and (2). The steady-state temperature \(\Delta T_\text {ss}\) of the three tissue types, i.e., the cerebellum, cerebrum, and brain stem, is tested for laser powers from 0.5 to 1.5 W, with 0.25 W steps and the results are shown in Fig. 3b, the error bars are weighted standard deviations of tissues from 5 different animals and the solid lines are linear fits to the data. The observed temperature increases are—like the results obtained for the artificial tissue—linearly proportional to the laser power, P. For cerebrum, cerebellum and brain stem, we found heating rates to be (\(8.2\pm 0.2\)) K/W, (\(8.4\pm 0.1\)) K/W, and (\(5.6\pm\) 0.1) K/W, respectively. Notably, the heating rate of brain stem is lower (\(p<<0.05\)) than the other two types of brain tissues, hence, brain stem heats significantly less when irradiated. Cerebrum and cerebellum are found to exhibit very similar heating rates (\(p=0.14\)). Furthermore, the heating rates of all types of tested brain tissues are larger than for the investigated phantom tissue, which is (\(1.4\pm\)0.3) K/W (Fig. 2b).
Plasmonic photothermal heating of brain tissue
To investigate the heating of laser-irradiated brain tissues with plasmonic nanoparticles, we first injected AuNS into the tissue. However, it was challenging to distribute the injected particles and we did not measure any significant difference in \(\Delta T_\text {ss}\) with or without AuNSs. Probably, this was due to the very inhomogenous distribution of the AuNSs in the tissue. To obtain a more homogeneous distribution of nanoparticles in the samples, we instead homogenized the brain tissue using a centrifugal stirrer (blender) before transferring the tissue to the cuvettes. Even though tissue homogenization disrupts the cell, membrane sheets, proteins, and ribonucleic acids remain intact (Goldberg 2014). The results of these experiments are shown in Fig. 4, which, for all three brain tissue types, displays the equilibrium temperature \(\Delta T_\text {ss}\), with nanoparticles (+AuNSs and +AgNP) and the control injected with the same volume of saline buffer (+PBS). Data point represents 4 or 5 independent experiments of each tissue type. Each sample is a mixture of tissue from two animals injected with either AuNS or AgNP to a final density of 5.8 \(\upmu\)g/ml. As expected, we found that the presence of plasmonic particles significantly augment \(\Delta T_\text {ss}\) compared to the control (+PBS) for all investigated tissue types. It is worth noticing that even though AgNPs were found to heat more efficiently than AuNSs in the artificial tissue, it is the other way around for the photothermal heating in porcine brain tissue. This is suggestive of a dramatic denaturing of the AgNP in the cerebral tissue. Therefore, an appropriate coating is needed to raise the structural stability in vivo (Espinosa et al. 2018). Still, however, for all tissue types, the effect is small compared to the experiments performed in artificial tissue where we found \(\Delta T_\text {ss}>4\) K (Fig. 2b). One obvious difference between the brain tissue and the artificial tissue, i.e., agarose, is the transparency. While the first is very dense, the artificial tissue is just slightly opaque and, hence, lets the majority of the laser light pass through the sample. To investigate this further, we measured the intensity loss in the tissue.
Absorbance spectroscopy in brain tissue
With photospectrometry in the ultraviolet to near-infrared range (UV–Vis–NIR), the absorbance of the homogenized porcine brain was evaluated. The absorbance is a dimensionless measure of the concentration-dependent attenuation or the loss of laser intensity in the tissue. To ensure accuracy, the absorbance was kept below 1 by diluting the homogenized brain tissue in a saline solution (PBS). UV–Vis–NIR spectra for the three tissue types can be seen in Fig. 5. Although the spectra have similar form, the magnitude of absorbance varies. The high absorbance of the brain stem is contradicted by the heating properties illustrated in Fig. 3, where the brain stem consistently shows a lower increase in temperature when irradiated by a laser. Since the brain stem does not absorb as much light as the other examined brain parts, scattering is suspected to be the main contributor to the measured absorbance. The curves in Fig. 5 show no signs of the absorption peaks outside of the biological window and no signs of an absorption peak when adding highly absorbent AuNSs (Additional file 2: Fig. S1). This is in agreement with the conclusion that the absorption is dwarfed by scattering.