Radiofrequency Hyperthermia of Cancer Cells Enhanced by Silicic

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Article Cite This: ACS Omega 2019, 4, 10662−10669

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Radiofrequency Hyperthermia of Cancer Cells Enhanced by Silicic Acid Ions Released During the Biodegradation of Porous Silicon Nanowires Maxim Gongalsky,*,†,# Georgii Gvindzhiliia,†,# Konstantin Tamarov,†,‡ Olga Shalygina,† Alexander Pavlikov,† Valery Solovyev,§ Andrey Kudryavtsev,§ Vladimir Sivakov,∥ and Liubov A. Osminkina*,†,⊥ Downloaded via 5.8.47.119 on July 18, 2019 at 05:55:28 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.



Department of Physics, Lomonosov Moscow State University, Leninskie Gory 1, 119991 Moscow, Russia University of Eastern Finland - Kuopio Campus, Yliopistonranta 1, 70210 Kuopio, Finland § Institute of Theoretical and Experimental Biophysics, Russian Academy of Science, Pushchino, 142290 Moscow Region, Russia ∥ Leibniz Institute of Photonic Technology, Jena 07745, Germany ⊥ Institute for Biological Instrumentation of Russian Academy of Sciences, Pushchino 142290, Russia ‡

S Supporting Information *

ABSTRACT: The radiofrequency (RF) mild hyperthermia effect sensitized by biodegradable nanoparticles is a promising approach for therapy and diagnostics of numerous human diseases including cancer. Herein, we report the significant enhancement of local destruction of cancer cells induced by RF hyperthermia in the presence of degraded low-toxic porous silicon (PSi) nanowires (NWs). Proper selection of RF irradiation time (10 min), intensity, concentration of PSi NWs, and incubation time (24 h) decreased cell viability to 10%, which can be potentially used for cancer treatment. The incubation for 24 h is critical for degradation of PSi NWs and the formation of silicic acid ions H+ and H3SiO4− in abundance. The ions drastically change the solution conductivity in the vicinity of PSi NWs, which enhances the absorption of RF radiation and increases the hyperthermia effect. The high biodegradability and efficient photoluminescence of PSi NWs were governed by their mesoporous structure. The average size of pores was 10 nm, and the sizes of silicon nanocrystals (quantum dots) were 3−5 nm. Degradation of PSi NWs was observed as a significant decrease of optical absorbance, photoluminescence, and Raman signals of PSi NW suspensions after 24 h of incubation. Localization of PSi NWs at cell membranes revealed by confocal microscopy suggested that thermal poration of membranes could cause cell death. Thus, efficient photoluminescence in combination with RF-induced cell membrane breakdown indicates promising opportunities for theranostic applications of PSi NWs.

1. INTRODUCTION

In order to improve RF-based methods, several contrast agents (for imaging) or RF sensitizers (for therapy) have been proposed.9 Most of them were gold nanoparticles due to their relatively good biocompatibility10 and strong interaction with RF electromagnetic fields. Gold nanoparticles enhanced both hyperthermia9,11 and RF ablation.12 However, the efficiency of such contrasts for RF methods13 and their possible toxicity issues14 are still under discussion. Recently, silicon nanoparticles (SiNPs) were proposed as alternative sensitizers of RF hyperthermia.15 RF treatment in the mild regime (hyperthermia without ablation) led to significant suppression of tumor growth due to enhancement by SiNPs absorbed in the tumor. The mechanisms of RF sensitizing by SiNPs were previously described in detail.16 In contrast to metal nanoparticles, SiNPs are highly biocompatible17 and biodegradable.18,19 Porous SiNPs were successfully

Application of radiofrequency (RF) electromagnetic radiation is a promising approach for therapy and diagnostics of numerous human diseases including cancer. The most common method that uses RF in oncology is RF ablation, which allows the elimination of malignant tumors with minimum invasion in contrast to a conventional surgery.1,2 Recently, RF ablation demonstrated one of the best results among all ablation surgery techniques, that is, prolongation of the overall survival of patients with highly aggressive tumors (for example, pancreatic cancer) from 11 to 20−25 months.3 Unresectable tumors can be treated by RF hyperthermia, which usually goes along with chemotherapy4,5 or gene therapy6 and improves the efficiency of the combined treatment. In addition to therapeutic modalities, the RF imaging system was recently applied to an angiography in vivo study.7 The setup used conductivity differences of tissues8 and demonstrated high imaging depth, but its sensitivity was limited due to the absence of efficient contrast agents.7 © 2019 American Chemical Society

Received: April 10, 2019 Accepted: June 4, 2019 Published: June 19, 2019 10662

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utilized for drug delivery20 and sensitizing of light and ultrasound.21 This allows multimodal applications of SiNPs desirable for combined anticancer therapy. Diagnostic modalities of nanoparticles could be combined with therapy in the framework of the so-called theranostic approach. For instance, nanoparticles can be applied as contrast agents for magnetic resonance imaging (MRI), fluorescent, ultrasonic, and optoacoustic imaging.22 Fluorescent agents are of greatest interest along with MRI agents.22 Porous SiNPs formed from porous silicon contain lots of silicon nanocrystals, which can be described as quantum dots if their diameter is less than 5 nm.23 These quantum dots exhibit efficient photoluminescence (PL) with a quantum yield of up to 20%.24,25 Emission spectra partially overlay with the human body transparency window, thus allowing in vivo fluorescence imaging. 26 The sensitivity of the bioimaging can be dramatically improved by the application of the time-gated regime.27 Typically, electrochemical etching of monocrystalline silicon wafers is utilized to obtain porous SiNPs, but there are some drawbacks. First of all, there is always a trade in between the size of the pores and quantum yield of the material. Therefore, the formation of highly luminescent SiNPs with high drug payloads requires additional chemical treatment.25,28 The second disadvantage is their spherical shape, which is vulnerable for phagocytosis29 and therefore exhibits shorter blood circulation time and worse uptake in tumors by comparison with high-aspect-ratio “worm-like” nanoparticles.30 From that perspective, metal-assisted chemical etching (MACE) allows obtaining highly photoluminescent porous silicon nanowires (PSi NWs), ready for efficient drug loading and delivery in a rapid synthesis procedure.31,32 Efficient photoluminescence (PL) of PSi NWs has the same origin and properties as in conventional porous silicon.33,34 However, PSi NWs have a mesoporous structure with pore sizes of 2−10 nm well-suitable for drug loading,35 reasonable PL quantum yields, and high aspect ratios, which are required for successful theranostic applications. The best policy to avoid unpredictable long-term toxicity of nanoparticles is to make them biodegradable. High biodegradability of SiNPs is another advantage for potential theranostic applications, and it was demonstrated by several research groups.19,26,36,37 The mechanism of biodegradation is the dissolution of silicon and surface silica into silicic acid (H4SiO4), which dissociates to H+ and H3SiO4− ions. These ions are harmless for live systems in reasonable concentrations and can be easily excreted via renal pathways. Biodegradation of PSi NWs was also demonstrated in in vitro experiments.38 However, the influence of the biodegradation processes on RF sensitizing properties of PSi NWs is still unknown. From this point of view, the present work is aimed to fill this gap and to improve the efficiency of nanoparticle-assisted RF hyperthermia.

Figure 1. (a,b) SEM images of PSi NW layer with different magnifications; (c,d) TEM images of detached PSi NWs with different magnifications. (e) Size distribution of nc-Si in single PSi NW obtained from the dark-field TEM image (Figure S3). (f) Pore size distribution of PSi NWs obtained from the desorption branch of N2 sorption isotherm.

from the substrate and partial fracturing of them, which resulted in the formation of individual nanowires as shown in the transmission electron microscopy (TEM) image in Figure 1c. The length of the PSi NWs after detachment was about 1− 2 μm, while a small fraction of submicrometer PSi particles was also present (details in the Supporting Information, Figure S2). Some of the PSi NWs may agglomerate; however, the typical size of agglomerates does not exceed 1−2 μm according to dynamic light scattering measurements (not shown here). This does not lead to a significant increase of cytotoxicity especially when taken into account the biodegradability of PSi NWs. Figure 1d shows the TEM image of the detailed structure of an individual PSi NW, which consists of a number of interconnected approximately spherical silicon nanocrystals (nc-Si) separated by pores. Each nc-Si creates a diffraction pattern, which was used for visualization of nc-Si in dark-field TEM images (see Figure S3). This data was used for the calculation of nc-Si size distribution shown in Figure 1e. The mean diameter dTEM was found to be 4.5 nm with a dispersion of equal to 3 nm, while the most probable size (maximum of distribution) of nc-Si was 3 nm. The relatively big dispersion points to the wide nc-Si size distribution, which means heterogeneity of their properties and the presence of nonuniform broadening effects. According to BJH calculations, the porosity of PSi NWs was about 67%, which corresponded to pore sizes of 3−20 nm (shown in Figure 1f). This

2. RESULTS AND DISCUSSION Figure 1a shows the scanning electron microscopy (SEM) micrograph of the layer of PSi NWs with a length of about 7 μm and NW diameter in the range from 100 to 300 nm (see Figure S1 in the Supporting Information), realized by MACE of the crystalline silicon (c-Si) wafer. The SEM image with higher magnification in Figure 1b reveals the porous structure of the PSi NWs. Mechanical scratching of PSi NW layers led to detachment of the NWs 10663

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Figure 2. Biodegradation study of PSi NWs after 1 day of incubation in H2O and PBS. (a) Top row: initial suspensions, bottom row: after 1 day of incubation at 37 °C; left column: digital photo of the suspensions, right column: photoluminescence excited by the UV lamp. (b) PL and (c) Raman spectra of PSi NW suspension before and after 1 day of incubation in H2O and PBS.

Figure 3. Relative temperature elevation of PSi NW suspension ΔT, that is, (a) temperature difference between degraded (after 1 day) and initial PSi NWs during RF heating under the same conditions. Blue and red curves correspond to incubation in H2O and PBS at 37 °C, respectively. (b) Schematic view of RF heating mechanism of the initial (left) and partially degraded after 1 day of incubation (right) PSi NW. Green and yellow circles represent cations and anions oscillating in the applied RF field, respectively.

PBS leads to the dissolution of nc-Si themselves, and the PL intensity decreases. PL of nc-Si is governed by the quantum confinement effect of excitons;24 therefore, the position of their emission band is determined by diameters of nc-Si according to the empirical equation

potentially allows an efficient payload of drugs into the pore network of PSi NWs.35 The results of the biodegradation of PSi NWs in H2O and in PBS at 37 °C are shown in Figure 2. The photographs in Figure 2a present the vials with initial 0.2 mg/mL suspensions of Si NWs and after 1 day of degradation in H2O and in PBS. The dissolution rate of PSi NWs in H2O (left vial on each photograph) was found to be too small to exhibit a significant visible effect after 1 day of incubation. Moreover, PSi NW photoluminescence (PL) enhancement was observed. At the same time, for PSi NWs incubated in PBS (right vial on each photograph), the dissolution is clearly visible; that is, the suspension became almost completely transparent. The higher dissolution rate of the PSi NWs in PBS can be explained by the higher ionic strength corresponding to the hydrodynamic activity of dissolved ions, the concentration of which was 6 orders of magnitude higher than it is in deionized water. The visual PL images of the vials are in good agreement with PL spectra presented in Figure 2b. The spectra show 3-fold enhancement of PL after 1 day of storage in H2O, which can be explained by additional activation of PL due to the slight oxidation of the PSi NW surface.39 Such oxidation results in passivation of nonradiative recombination centers, that is, dangling bonds, while the average size of nc-Si is not significantly affected.40 On the contrary, strong oxidation in

dPL

ij 3.73 yz zz = jjjj z j ΔEg zz k {

0.72

(1)

where ΔEg is the increase of the effective band gap due to quantum confinement by comparison with the bulk silicon band gap Eg0 = 1.12 eV. From the peak position of PL shown in Figure 2b, eq 1 gives the nc-Si sizes dPL close to 3.3 nm, which is in good agreement with TEM data (Figure 1e). A decrease in the PL intensity and its blue shift during the PSi NW incubation in PBS indicate a decrease of the ns-Si size due to the biodegradation processes. Similar conclusions can be made from Raman studies of PSi NWs presented in Figure 2c. The distinctive Raman band near 520 cm−1 attributed to scattering on longitudinal optical phonons in Si41 allows monitoring biodegradation of PSi NWs. The intensity of the band is proportional to the concentration of silicon, whereas the band position is governed by the diameter of nc-Si dRS according to the equation41 10664

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i 52.3 yz zz dRS = 0.543jjj k Δω {

Article 0.63

(2)

where Δω is a Raman band shift from the bulk c-Si position of 520.5 cm−1. The mean diameter dRS was about 5.5 nm for initial nc-Si in PSi NWs, which is slightly higher than the corresponding value of dPL. This discrepancy in sizes was attributed to the absence of bigger Si nanocrystals, which do not contribute to the PL spectrum. Incubation of PSi NWs in water led to a further shift of the band position and the broadening of the band corresponding to a slight shrinking of the nc-Si sizes, while the intensity of the band did not change dramatically. The calculated dRS after 1 day of incubation in water was about 3.3 nm, which is in good agreement with dPL, and indicates also the slight degradation of nc-Si and achievement of quantum confinement conditions. On the contrary, incubation of PSi NWs in PBS led to complete vanishing of the Raman Si band that points to the almost total dissolution of PSi NWs. The effect of PSi NW biodegradation on radiofrequency (RF) heating was investigated using a clinically approved RF apparatus and noncontact infrared thermometer. Figure 3a shows a relative temperature elevation ΔT, that is, the temperature difference between suspensions of degraded (after 1 day) and initial PSi NWs during RF heating under the same conditions, in H2O (blue curve) and PBS (red curve). Raw temperature dependences are presented in Figure S4. The absence of the dissolution effect was clearly visible when incubating PSi NWs in H2O, and the low dissolution rate of PSi NWs in H2O resulted in a low-temperature difference below 0.5 K. On the contrary, the high dissolution rate of PSi NWs in PBS resulted in formation of highly concentrated silicic acid (dissociated to the ions) and led to a significant increase of the heating rates of suspensions (0.2 K/s). The process of PSi NW dissolution in aqueous media with the formation of silicic acid and its dissociation into ions is described by the equation42 SiO2 + 2H 2O → H4SiO4 ⇔ H+ + H3SiO4 −

Figure 4. In vitro study of PSi NWs. (a,b) Confocal images of PSi NWs incubated with Hep2 cells for 4 and 24 h, respectively. (c,d) Hep2 viability vs RF irradiation time and concentration of PSi NWs. The bars show mean ± SD (n = 4). The levels of significance between the control and treated groups were set at a probability of ****p < 0.0001.

2). Thus, the efficient PL provides diagnostic modality of PSi NWs. The relatively large length of PSi NWs (1−2 μm) partially prevents them from penetrating through the cell membrane; thus, the majority of PSi NWs are localized in the vicinity of the membranes for both 4 and 24 h of incubation. The confocal z-scan image stack (Figure S6) shows negligible penetration of PSi NWs through the cell membrane. However, some of PSi NWs may be internalized in endosomal compartments of cells.43 The endosomal media is usually acidic with low pH, which may significantly change biodegradation of inorganic nanowires.44 To clarify this point, we investigated biodegradation of PSi NWs in simulated acidic conditions, that is, an aqueous solution of citric acid (150 mM) with an equal ionic strength to PBS. The data are shown in the Supporting Information. Figures S7 and S8 demonstrate huge deceleration of biodegradation in acidic conditions. So, Figure S7 shows a slight shift of the Si Raman band, in contrast, to complete vanishing of the band after degradation in PBS. Figure S8 shows no significant effect (within the margins of errors) of PSi NW incubation in citric acid solutions on the RF heating rate. Therefore, the influence of RF activation internalized in the endosomal compartment of the cells PSi NPs can be considered as negligible. Figure 4c,d demonstrates the effect of RF irradiation on cell viability. Blue bars correspond to the control experiment, that is, cells without PSi NWs. Pink, green, and red bars correspond to PSi NW concentrations of 0.05, 0.1, and 0.2 mg/mL added to cell cultures, respectively. Note that nontoxic PSi NW concentrations were chosen in this experiment (see the Supporting Information, Figure S9). RF irradiation exposure values were 0, 5, 10, 20, and 30 min (groups of barsfrom left to right). The combined action of RF and PSi NWs after 4 h of incubation with cells did not significantly influence cell viability (Figure 4c). The majority of cells maintained their viability

(3)

A schematic view of the mechanism of PSi NW heating under RF irradiation is shown in Figure 3b. The double-sided arrow displays the oscillations of ions under the action of the RF field, leading to energy dissipation and Joule heating of the suspension: green circles are cations (H+), and yellow circles are anions (H3SiO4−). The concentration of ions is higher for the suspension of partially degraded PSi NWs after 1 day of its incubation in PBS, which causes greater heating of the suspension in comparison with the initial ones. In water, the degradation of PSi NWs is slow compared to that in PBS, and the difference in the heating of the initial suspensions and after 1 day of incubation is imperceptible. These results are consistent with the suspension conductivity measurement presented in the Supporting Information (Figure S5 and Table S1). Figure 4 shows the results of in vitro experiments with Hep2 cells exposed to the applied RF field in the presence of PSi NWs. Figure 4a,b presents fluorescent confocal images of the cells after 4 and 24 h of incubation with PSi NWs, respectively. Blue, green, and red colors correspond to cell nuclei, cytoplasm, and PSi NWs, respectively. Note that the red color of PSi NWs is explained by their natural label-free PL of quantum-confined excitons in silicon nanocrystals (see Figure 10665

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after 24 h of incubation, the PSi NWs are attached to the cell membrane. Biodegradation of PSi NWs during 24 h incubation with cells leads to a decrease in the size of silicon nanocrystals and the formation and dissociation of silicic acid. A large number of ions, in turn, lead to large heating of the suspensions (represented as a red cloud) due to the release of Joule heat when exposed to an RF field. Both measurement and calculation of local ion concentration are difficult to perform; however, overall concentration of ions can be estimated as less than 15 mM for the completely degraded 0.2 mg/mL PSi NW suspension. The fracturing of the cell’s membrane in the vicinity of “hot” PSi NWs is shown as breaks of the black line.

after 5 and 10 min of RF irradiation independent of PSi NW concentration. That points to the mild regime of RF treatment, which is potentially applicable to in vivo experiments and clinical trials. However, longer RF treatment led to significant temperature elevation and hyperthermia causing cell death and viability drop down to 10−20%. Thus, sensitizing of the RF field by PSi NWs does not show any additional effect in in vitro experiments after 4 h of their incubation with cells. The effect of RF sensitizing by PSi NWs was observed after 24 h of incubation. Figure 4d shows strong concentration dependency for cell cultures exposed to RF irradiation for 10 min. The viability of control cells (without PSi NWs) was approximately 80%, similar to the case of 4 h of incubation. Addition of PSi NWs in low concentration (0.05 mg/mL) led to a significant viability drop to 40%, while the further increase of PSi NW concentration led to the almost complete destruction of cells (viability was under 15%). With an increase in irradiation times, hyperthermic effects of RF were predominant, and the difference in cell survival without and in the presence of PSi NWs was insignificant. Therefore, for successful application of RF therapy strict conditions for RF intensity and exposure time, the concentration of PSi NWs and time of incubation (time after injection of PSi NWs in potential clinical trials) must be fulfilled. Otherwise, the treatment can be weak to affect any dangerous cancer cells or too strong to keep healthy cells without PSi NWs alive. However, the precise application of RF treatment and PSi NWs allows 5-fold enhancement of cancer cell destruction by comparison with cells without PSi NWs. The schematic representation of the influence of PSi NW biodegradation on the effectiveness of RF sensitizing and cancer cell destruction is shown in Figure 5. First, PSi NWs are

3. CONCLUSIONS It has been shown that radiofrequency hyperthermia sensitized with porous silicon nanowires can be significantly improved by fine-tuning the processing parameters, including the radiofrequency exposure time, intensity, concentration of nanoparticles in cell cultures, and incubation time. The mechanism of the improvement is based on the formation of silicic acid ions during the nanowire biodegradation process in the cellular medium. These ions change the electrolytic properties of PSi NW suspensions, which in turn lead to higher Joule heating and temperature increase under RF radiation followed by cell death. Localization of the silicon nanowires in the vicinity of the cell membranes was demonstrated by confocal microscopy facilitated by intrinsic photoluminescence of silicon quantum dots inside the nanowires. Thus, the combination of radiofrequency thermal therapy sensitized by PSi NWs and photoluminescent properties opens prospects to their use in clinical anticancer theranostics. 4. MATERIALS AND METHODS 4.1. Preparation of Porous Silicon Nanowires. PSi NWs were obtained by MACE of a highly doped (100)oriented single-crystalline silicon wafer (0.001 Ω cm). At the first step, the Si wafer was immersed in a mixture of 0.01 M AgNO3 and 5 M HF in a volume ratio of 1:1 for 15 s. As a result, Ag nanoparticles were sedimented on the surface of the Si wafer. Then the Si wafer was placed into a mixture of 5 M HF and 30% H2O2 in a volume ratio of 10:1 for 1 h. The layer of PSi NWs occurred because of MACE. Then the samples were rinsed three times with water and then for 10 min in 50% HNO3 to remove Ag. Then the samples were rinsed again three times in water and dried at 50 °C in air. The resulting PSi NW layer was scrubbed from the surface, and suspensions with a concentration of 0.2 mg/mL were made in H2O, PBS, and monohydrated citric acid (150 mM, pH = 2). The necessary amount of PSi NWs was weighed with 0.05 mg accuracy before the formation of suspensions. 4.2. Characterization of Porous Silicon Nanowires. SEM images were obtained by means of a field emission scanning electron microscope (Carl Zeiss ULTRA 55 FESEM) operated at an acceleration voltage of 10 kV. TEM images were obtained with an LEO 912 AB Omega transmission electron microscope (Zeiss, Oberkochen, Germany) operated at an acceleration voltage of 120 kV. Size distributions (in Figure 1e−g) were calculated from a TEM image using ImageJ software. The image was modified by contrast enhancement in GIMP software, and then dark/bright spots corresponding to pores/crystallites were outlined and

Figure 5. Schematic representation of the mechanism of RF sensitizing by PSi NWs. Reddish glow near PSi NWs represents RF-induced heating. Small green and yellow circles are silicic acid cations and anions, respectively. PSi NWs on the left represent initial particles with thick silicon walls, and PSi NWs in the vicinity of the cytoplasmic membrane (colored black) represent highly biodegraded particles with thin silicon walls after 24 h of incubation. The fracturing of the membrane in the vicinity of “hot” PSi NWs is shown as breaks of the black line.

localized near or on the cell membrane after 4 h of incubation. Small green and yellow circles in the figure are silicic acid cations and anions, respectively. Silicon nanocrystals in PSi NWs are quite large and are represented in gray. Thus, slight degradation of PSi NWs and a small amount of ions lead to slight heating of the suspension, which is represented as a pink cloud. Such low heat does not lead to cell destruction. Then, 10666

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fitted by ellipses. Each ellipse gave us two values, that is, major and minor axes, which were both used for the calculation of pore/crystallite diameter distribution (Figure 1g) or used separately for calculations of length and width of detached PSi NWs, respectively (Figures S1 and S2). For the latter, only the nonspherical particles were taken into account. Backscattered Raman spectra were measured using a Horiba HR 800 micro-Raman spectrometer under excitation by the 6 mW He−Ne laser (wavelength 632.8 nm). The spectral resolution was 0.5 cm−1. PL of PSi NWs was measured by a SOLAR TII MS 750 spectrometer equipped by a CCD camera with a Si-based Hamamatsu HS 101H-HR matrix. PL was excited by a He−Cd laser with a wavelength of 325 nm. The pore sizes, surface area, and pore volumes were determined with N2 adsorption/desorption (Quantachrome NOVA 4200e, Quantachrome Instruments). The surface area was calculated using the BET theory (Brunauer, Emmett, and Teller) from the adsorption branch,45 and the pore size distribution was calculated from the desorption branch using the BJH theory (Barrett, Joyner, and Halenda).46 4.3. Heating of PSi NW Suspensions in RF Field. For heating of PSi NW suspensions, a clinically approved RF generator “MedTeKo UHF60” was used. The frequency was 27.12 ± 0.16 MHz. The power of the RF source was 60 W, which corresponds to a power density of 1.5 W/cm3. A cuvette with a suspension was placed between round electrodes with a diameter of 36 ± 5 mm, and the distance between them was 4 cm. The temperature was measured by a fast noncontact infrared thermometer with 0.2 °C accuracy. The electrical conductivity of the solutions was measured by using an HP 4192A LF impedance analyzer in the range of 5 kHz to 13 MHz. 4.4. In Vitro Experiments. Hep2 cells were routinely cultured in Dulbecco’s modified Eagle’s medium (DMEM, PanEco, Russia) supplemented with 10% fetal bovine serum (FBS, Gibco, USA) and 1% antibiotic (gentamycin, DHF OAO, Russia) in 75 cm2 flasks in a CO2 incubator. Before the experiment, a total of 104 cells per well in 100 μL of culture medium were seeded in a 96 well plate and were allowed to attach for 20 h. Then DMEM was replaced with L-15 (Sigma, US) media, which contained the predetermined concentrations of PSi NWs, and the cells were incubated for 4 or 24 h to allow PSi NWs to dissolve and change the heating rate. The necessary amount of PSi NWs was added to L-15 media to reach a concentration of 0.2 mg/mL. Since the RF generator was too large to be placed into the incubator, a different chamber was devised where the 96 well plate and RF electrodes below and above the plate were placed. The temperature inside the chamber was kept at 37 °C. The L15 media was needed to ensure the constant pH value in the cell media in the absence of CO2 in the chamber. The cells were irradiated for 5, 10, 20, and 30 min with 40 W power, after which L-15 was replaced with fresh DMEM and cells were additionally incubated for 8 h before the viability measurement. The measurement was done with a CellTiter Glo (Promega) assay according to the manufacturer’s instructions. The obtained results were analyzed with twoway ANOVA with Bonferroni multiple comparison correction. The same cell line was used for confocal imaging. After addition of PSi NWs, 3 μg/mL of Calcein-AM purchased from Sigma and 5 μg/mL of bisbenzimide H 33342 (Hoechst) purchased from Calbiochem were added and incubated for 1 h in the presence of 5% CO2. Confocal images were obtained by

means of a Leica confocal microscope. Excitation was provided by laser diodes with wavelengths of 405 and 488 nm. Emission was measured at 460 and 550 nm.



ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.9b01030.



General view SEM images of PSi NWs; length and width distributions of PSi NPs; dark-field TEM image of PSi NWs and outlines used for size distribution calculation; raw temperature dependences on RF heating time for all PSi NW suspensions; frequency impedance dependences for PSi NW suspensions and table of specific DC conductivities; full z-scan family of images; experiment with incubation of PSi NPs in citric acid solutions; cytotoxicity study data (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (M.G.). *E-mail: [email protected] (L.A.O.). ORCID

Maxim Gongalsky: 0000-0002-7069-4234 Liubov A. Osminkina: 0000-0001-7485-0495 Author Contributions #

M.G. and G.G. contributed equally.

Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS The study was supported by the Russian Science Foundation grant no. 17-72-10200 (everything except RF phantom experiments) and by the “UMNIK” program of the Foundation for Assistance to Small Innovative Enterprises in Science and Technology, project no. 13098GU/2018 (RF phantom experiments).



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DOI: 10.1021/acsomega.9b01030 ACS Omega 2019, 4, 10662−10669

ACS Omega

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DOI: 10.1021/acsomega.9b01030 ACS Omega 2019, 4, 10662−10669