A novel cascaded energy conversion system inducing efficient and precise cancer therapy.

Cancer therapies based on energy conversion, such as photothermal therapy (PTT, light-to-thermal energy conversion) and photodynamic therapy (PDT, light-to-chemical energy conversion) have attracted extensive attention in preclinical research. However, the PTT-related hyperthermia damage to surrounding tissues and shallow penetration of PDT-applied light prevent further advanced clinical practices. Here, we developed a thermoelectric therapy (TET) based on thermoelectric materials constructed p-n heterojunction (SrTiO3/Cu2Se nanoplates) on the principle of light-thermal-electricity-chemical energy conversion. Upon irradiation and natural cooling-induced the temperature gradient (35-45 oC), a self-build-in electric field was constructed and thereby facilitated charges separation in bulk SrTiO3 and Cu2Se. Importantly, the contact between SrTiO3 (n type) and Cu2Se (p type) constructed another interfacial electric field, further guiding the separated charges to re-locate onto the surfaces of SrTiO3 and Cu2Se. The formation of two electric fields minimized probability of charges recombination. Of note, high-performance superoxide radicals and hydroxyl radicals' generation from O2 and H2O under catalyzation by separated electrons and holes, led to intracellular ROS burst and cancer cells apoptosis without apparent damage to surrounding tissues. Construction of bulk and interfacial electric fields in heterojunction for improving charges separation and transfer is also expected to provide a robust strategy for diverse applications.

Introduction

Very recently, with the rapid development of nanotechnology and nanomedicine, energy-conversion material-mediated therapies have aroused comprehensive attention due to their non-invasive feature and reliable therapeutic effect. Among them, photothermal therapy (PTT) based on light-thermal conversion and photodynamic therapy (PDT) based on light-chemical energy conversion are the most representative energy-conversion therapies [[1], [2], [3], [4], [5]]. To achieve a high anticancer efficiency, two essential criteria of traditional PTT and PDT should be considered, including high light-thermal or light-chemical energy conversion performance of the employed agents and long light penetration with minimized tissue scattering and absorption [[6], [7], [8], [9]]. Typically, energy conversion efficiency is the critical factor for an eligible nanomedicine. For example, it is quite necessary for a photothermal agent to increase the temperature of the tumor site above 50 °C for achieving a desired therapeutic outcome. Additionally, in order to improve light penetration, great endeavors have been devoted to develop the second near infrared ray-based materials (NIR-II) in the spectra range of 1000–1350 nm, which possesses deeper tissue-penetration, reduced light scattering, and higher skin permissible exposure (MPE) than that of NIR-I and visible light [[10], [11], [12], [13], [14]]. Nevertheless, only limited examples of NIR-II photothermal agents and negligible NIR-II photodynamic agents have been reported. Besides, the tissue-penetration of NIR-II light was greatly affected by the strong absorbance band of water overtone, causing potential thermal damage to normal organs and tissues [[15], [16], [17], [18]]. Therefore, despite these great advances achieved to date, most of the existing PDTs are limited to superficial treatments, and most of the existing PTTs are not specifically related to cancer-associated events. That is, although light irradiation could target the tumor site, hyperthermia induced by conventional photothermal agents or ambient fluid body, would randomly propagate and diffuse to the surrounding normal tissues and organs, and thus results in the treatment-related toxicity and side effects, which are the major obstacle preventing further advanced clinical practice of PTT.

Over the last few decades, thermoelectric (TE) materials, through converting thermal to electricity via electron-hole pairs separation under temperature gradient-induced the build-in thermoelectric field, have attracted tremendous attention worldwide in materials science and solid-state physics, due to their wide application in Peltier cooling and waste energy harvesting [[19], [20], [21], [22], [23], [24], [25]]. Further analysis of the mechanism of TE materials shows the separated electron-hole pairs under build-in thermoelectric field demonstrating great potentials in catalyzing reactive oxygen species (ROS) generation in cushy conditions, similar with the mechanisms of photodynamic therapy and piezocatalytic therapy [26,27]. In addition, compared with PTT-generated hyperthermia, the ROSs have much shorter lifetime and propagation distance in vivo, presumably guaranteeing much less treatment-related side effect or toxicity on surrounding normal tissues and organs. Although TE generators have grown into superstars in the application fields of energy and environment, it is still an infant in biomedical fields. Typically, the thermoelectric figure of merit is the key to evaluate the efficiency of TE materials, ZT = S2σΤ/κ, where S, σ, Τ and κ represents Seebeck coefficient, electrical conductivity, temperature, and thermal conductivity, respectively [28,29]. Obviously, the thermoelectric efficiency not only relates to physicochemical properties of TE materials, such as Seebeck coefficient, electrical conductivity, and thermal conductivity, but it also has a linear correlation with the operating temperature, in which higher temperature endows more effective electron-hole pairs separation and higher thermoelectric efficiency, which is also the main obstacle for applications in biomedical fields of TE materials [30]. Based on our previous studies [[31], [32], [33], [34], [35], [36]], heterojunction construction, including p-n junction and Z scheme junction, has been demonstrated as an efficient strategy for improving electron-hole pairs separation and enhancing the catalytic efficiency. After contacting of p-type and n-type semiconductors, the interfacial electric field will be constructed, in which the separated electrons and holes in both semiconductors would transfer in the opposite direction and locate in different semiconductors [[37], [38], [39], [40]]. The unfavorable recombination of electron-hole pairs would be retarded, which is the key for conversion efficiency of photocatalysts, electrocatalysts, and TE materials.

Herein, for the first time, we developed a novel thermoelectric therapy (TET) based on light-thermal-electricity-chemical energy conversion, by employing SrTiO3 (n type) and Cu2Se (p type) to construct a p-n heterojunction, which is capable of dual independently targeted generating ROS under mild temperature gradient (from 35 °C to 45 °C). As shown in Scheme 1, by employing conventional two-step hydrothermal processes, SrTiO3/Cu2Se based p-n heterojunction was constructed. Under 808 nm laser irradiation and natural cooling, the electrons and holes in the bulk of SrTiO3 and Cu2Se voluntarily separate and migrate from the bulk to the surface under the driving force of the build-in thermoelectric field on the opposite directions. Additionally, the p-n heterojunction between SrTiO3 and Cu2Se constructs an interfacial electric field, and thus redistributes the surface electrons and holes to specific locations for reduction and oxidation reactions, respectively, which further restrains the undesired recombination of electrons-hole pairs in the bulk and on the surface of TE materials. Consequently, a ROS burst under mild temperature gradient and low concentration of TE materials was provided based on a thermoelectric effect. With the goal of complete remission of tumors and without recurrence, our work here presents a novel thermoelectric mechanism based on p-n heterojunction constructed TE generator, with dual independently targeted ROS bursts for efficient cancer therapy and with negligible side effects towards normal tissues. To be noted, we also anticipate the performances of such a p-n heterojunction-constructed TE generator in other settings of biomedical applications beyond cancer treatment.

Scheme 1

Schematic illustration of preparation and mechanism of SrTiO/CuSe p-n heterojunction based thermoelectric therapy (TET). SrTiO3/Cu2Se p-n heterojunction was fabricated through two steps hydrothermal process.

Materials and methods

Materials

Ti(OBu)4, Sr(NO3)2, NaOH, (CH2OH)2, Se, CuI, H2N(CH2)NH2, 9,10-anthracenedipropanoic acid (ABDA), [Ru(dpp)3]Cl2 (RDPP), H2O2 (30%), N-methyl-pyrrolidone (NMP), methylene blue (MB), 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB), and dihydrorhodamine 123 (DHR123) were supplied by Sigma-Aldrich.DSPE-PEG-Cy7 (MW: 5k) and DSPE-PEG (MW: 5k) were purchased from Nanocs Inc. PBS (pH 7.4 and 5.5), DMEM medium, RPMI medium, trypsin-EDTA, and fetal bovine serum (FBS) were purchased from Gibco Life Technologies.

Synthesis of SrTiO3/Cu2Se NPs

The SrTiO3/Cu2Se NPs based p-n heterojunction was prepared by simple two-step hydrothermal processes. First, SrTiO3 NPs were synthesized by a hydrothermal method using Teflon lined stainless steel autoclave containing the mixture of (CH2OH)2, NaOH, Sr(NO3)2, and Ti(OBu)4. After the steel autoclave was heated at 180 °C for 24 h, the obtained powder was under probe sonication–assisted liquid exfoliation in NMP for 5 h. Then, the solution was centrifuged at 3000 rpm for 5 min to obtain SrTiO3 NPs. For Cu2Se QDs coating, CuI and Se powders were placed in a Teflon lined stainless steel autoclave containing 30 mL ethylenediamine and prepared SrTiO3 NPs. After the steel autoclave was heated at 90 °C for 4 h, the precipitate was centrifuged at 3000 rpm and washed with distilled water for 3 times. Then, the prepared products were dried in vacuum at 50 °C for 4 h. The final black products (SrTiO3/Cu2Se NPs) were collected.

For surface modification, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-polyethyleneglycol (DSPE-PEG) was dissolved into the SrTiO3/Cu2Se NPs solution and stirred for 12 h after 30 min of sonication. Then the solution was centrifuged for 30 min at 5000 rpm with Amicon pipes (MWCO 100 kDa; Millipore) and the precipitates were washed 3 times to remove the residual DSPE-PEG. The same process was used for fluorescent modification of SrTiO3/Cu2Se NPs using DSPE-PEG-Cy7 in a dark environment.

Characterization

The zeta potential and corresponding size of SrTiO3/Cu2Se NPs were detected through Dynamic Light Scattering. The morphology of SrTiO3/Cu2Se NPs was observed with transmission electron microscopy (TEM, JEM-2100UHR, JEOL, Japan) and atomic force microscopy (AFM, FASTSCANBIO, Germany). Piezoresponse force microscopic (PFM) measurements were characterized by an AFM (NTEGRA, NT-MDT, Russian) equipped with a ferroelectric test system. The SrTiO3/Cu2Se NPs chemical constituents were detected by energy-dispersive X-ray spectroscope (EDS) (Inca X-MAX, Oxford, UK), X-ray photoelectron spectroscopy (XPS, ESCALAB 250Xi, Japan), and fourier transform infrared spectrophotometry (FTIR, Nexus 470, Nicolet, Madison, WI, USA). The SrTiO3/Cu2Se NPs chemical structures were characterized by employing X-ray powder diffraction (XRD, Bruker D8 multipurpose). As for thermoelectric properties, a laser flash method (LFA 457, NETZSCH) was utilized to measure thermal diffusivity (D), while a ZEM-3, ULVAC was used for the analysis of σ and S.

·OH production in vitro

The mixture of SrTiO3 NPs, Cu2Se QDs or SrTiO3/Cu2Se NPs and MB were prepared in PBS (pH 7.4) and stirred for 1 h in a dark environment at the corresponding final concentration of 0.050 mg/mL TE agents and 0.05 mg/mL MB, respectively. Then the mixture was irradiated with 808 nm laser (1 W/cm2) for 2.5 min and cooled naturally for 10 min. The diminished MB was determined by recording the absorption of the supernatant via UV–vis spectroscopy.

·O2− production in vitro

The mixture of SrTiO3 NPs, Cu2Se QDs or SrTiO3/Cu2Se NPs (0.05 mg/mL) and DHR123 (1 μL,1 mM) were formed in PBS (pH 7.4) and stirred for 1 h in a dark environment. Then the mixed solution was irradiated via 808 nm laser (1 W/cm2) for 2.5 min and natural cooling for 10 min. The changing fluorescence of DHR123 was recorded by fluorescence spectrophotometer.

Electron Paramagnetic Resonance (EPR) for ·OH and ·O2−in vitro

For further detection of the ·OH and ·O2− formation, SrTiO3/Cu2Se NPs (0.05 mg/mL) and 5,5-dimethyl-1-pyrroline N-oxide (DMPO) (0.1 mM) were fully mixed (solution for testing ·OH: DI water, solution for testing ·O2−: methanol). The respective signals were analyzed by Electron Paramagnetic Resonance (EPR).

Cytotoxicity of TE agents

MCF 7 and Hela cells were plated and cultured in 96-well microplates (37 °C, 5% CO2) for 24 h. Then the SrTiO3 NPs, Cu2Se QDs or SrTiO3/Cu2Se NPs at different concentrations (ranging from 0.025 mg/mL to 0.1 mg/mL) were mixed into the culture medium. Following 24h co-incubation, MTT assay (Life Technologies) was conducted to determine cell viabilities according to the manufacturer's instructions.

Production of ROS in cells

After 24 h of culture (37 °C, 5% CO2) in dishes, MCF 7 cells were incubated with the SrTiO3 NPs, Cu2Se QDs and SrTiO3/Cu2Se NPs (final concentration of 0.05 mg/mL) respectively for another 24 h. Next, the DCFH-DA solution (final concentration of 0.2 μM) was put into the above medium and incubated for 0.5 h. Following removing the culture medium and washing with PBS, the cells were illuminated for 2.5 min employing an 808 nm light with power 1 W/cm2 and cooled naturally for 10 min. The green fluorescence induced by ROS was detected by CLSM.

In vitro TET

MCF 7 and Hela cell lines were plated in 96-well plates and cultured for 24 h (37 °C, 5% CO2). Then the SrTiO3 NPs, Cu2Se QDs or SrTiO3/Cu2Se NPs at different concentrations (ranging from 0.025 mg/mL to 0.1 mg/mL) were mixed into the culture medium and incubated for another 24 h. Following culture medium removing and PBS washing, the cells were illuminated for 2.5 min employing an 808 nm light with power 1 W/cm2 and cooled naturally for 10 min. After 24-h culture, the cells were washed with PBS for several times. MTT assay was applied to detect cell viability.

Pharmacokinetic study

To conduct in vivo pharmacokinetic study, 200 μL of Cy7 functionalized SrTiO3/Cu2Se NPs (6 mg/kg) were i.v. injected in C57BL/6 mice. After different intervals, a microplate reader was utilized to test fluorescent intensity of Cy7 through the collected 20 μL blood.

In vivo imaging and biodistribution study

For the sale of fluorescence imaging and biodistribution study in vivo, 200 μL of Cy7 functionalized SrTiO3/Cu2Se NPs (6 mg/kg) were i.v. injected into mice bearing MCF 7 tumors. The Maestro2 In-Vivo Imaging System was employed to detect the fluorescent intensity of tumor and major organs 24 h post-injection.

In vivo TET

After the tumors had reached to ∼100 mm3, mice were randomly allocated into 6 groups, 5 mice in each group. 1) saline control, 2) SrTiO3/Cu2Se NPs, 3) SrTiO3 NPs + ΔT, 4) Cu2Se QDs + ΔT, 5) SrTiO3/Cu2Se NPs + ΔT, 6) G + ΔT, and 7) PTT (G, >55 °C). The injected doses of SrTiO3/Cu2Se NPs, SrTiO3 NPs, and Cu2Se QDs were 6 mg/kg. The ΔT means temperature gradient (35–45 °C) for 3 cycles inducing by 808 nm laser irradiation and natural cooling after 24 h post injection. The volume of tumors was monitored every two days lasting 14 days. All animal experiments were performed according to protocols approved by the Institutional Animal Care and Use Committee of Sun Yat-sen University (Guangzhou, China).

In vivo toxicity

C57BL/6 mice were i.v. injected with SrTiO3/Cu2Se NPs (10 mg/kg) in order for toxicity analysis in vivo. Major organs were collected and subjected to eosin (H&E) and hematoxylin staining 30 days postinjection. Then the immune response was analyzed through the i.v. injection of SrTiO3/Cu2Se NPs (10 mg/kg) into C57BL/6 mice. At 12 h and 24 h post-injection, ELISA was used to measure the concentrations of interleukin6 (IL-6), tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ). At day1, 7, and 14 after intravenous injection, urea nitrogen (BUN), creatinine (Cr), albumin (ALB), total protein (TP), aspartate alanine aminotransferase (ALT), and aminotransferase (AST) were detected in a whole blood panel.

Statistical analysis

Data statistics and statistical significance were calculated by using Graph Pad Prism 8.0 and Origin 9.0. And NPs biodistribution and tumor volume were analyzed via employing Microsoft Excel 2016.

Results and discussion

Preparation and characterization of SrTiO3/Cu2Se p-n heterojunction

In the first stage of this work, the SrTiO3/Cu2Se p-n heterojunction was synthesized through two steps of hydrothermal process. Detailly, as illustrated in Scheme 1, SrTiO3 nanoplates (NPs) (n type TE materials) were papered firstly following Cu2Se quantum dots (QDs) synthesis and in site coating on the surface of SrTiO3 NPs. After the hydrothermal process and liquid exfoliation, SrTiO3 NPs with a size of 110 nm and a polydispersity index (PDI) of 0.182 were analyzed by transmission electron microscopy (TEM) (Fig. 1a and Fig. S1). Then, Cu2Se QDs, p type TE materials, were synthesized and coated on the surface of SrTiO3 NPs, forming a novel p-n heterojunction-based TE generator. The successfully coated Cu2Se QDs was obviously observed in the TEM images of SrTiO3/Cu2Se NPs (Fig. 1b), in which uniform QDs decoration on the surface of SrTiO3 NPs was exhibited. The size and PDI of the prepared SrTiO3/Cu2Se NPs were appropriately increased to about 132 nm and 0.256 (Fig. S2). Additionally, the high-resolution transmission electron microscopy (HRTEM) images with clear interference fringe and d-spacing of 0.27 nm and 0.33 nm, corresponding to the plane of SrTiO3 NPs (Fig. 1c) and Cu2Se QDs (Fig. 1d), provided direct evidence for the successful fabrication of the heterojunction structure. Besides, atomic force microscope (AFM) was also applied to characterize the morphology of the fabricated SrTiO3/Cu2Se NPs. As shown in Fig. 1d and e, a rough surface was clearly observed, which was likely attributed to the coating of Cu2Se QDs. Calculation from the 2D and 3D AFM images of SrTiO3/Cu2Se NPs, the NPs displayed a planar size of about 132 nm and thickness of 50 nm. Given that almost all thermoelectric materials have a good piezoelectric property [[41], [42], [43]], we determined the piezoelectric properties of SrTiO3/Cu2Se NPs by a piezoresponse force microscope (PFM), using dual alternating current resonance tracking (DART) modes with the aim to expel the displacement contribution from electrostatic interaction and topographical crosstalk in mapping the local electromechanical properties. Fig. 1e–h exhibited the topographic, vertical piezoresponse amplitude, and phase images of the SrTiO3/Cu2Se NPs, respectively. The SrTiO3/Cu2Se NPs can be clearly detected in the topographic image with clear contrast in the amplitude and phase images. Fig. 1g exhibited the phase map of SrTiO3/Cu2Se NPs, which well matches the morphology map in Fig. 1e. Next, a surface potential of 30 mV of SrTiO3/Cu2Se NPs was observed in the piezoelectric potential map of SrTiO3/Cu2Se NPs (Fig. 1h) obtained by PFM in the darkness, which demonstrates its piezoelectric property and further testifies its thermoelectric property. To further confirm the successful fabrication and the composition of SrTiO3 NPs, Cu2Se QDs and SrTiO3/Cu2Se NPs, X-ray photoelectron spectroscopy (XPS) and X-ray diffractometry (XRD) were performed. In the XPS analysis (Fig. 2a), the specific peaks of Sr, Ti, O, and Cu, Se, were exhibited in their XPS spectra, respectively. Moreover, all these characteristic peaks were observed in the XPS spectrum of SrTiO3/Cu2Se NPs. For the XRD detection, as shown in Fig. 2b, the obtained SrTiO3 NPs, Cu2Se QDs, and SrTiO3/Cu2Se NPs exhibited high phase purity, which is evidenced from their XRD pattern. The XRD peaks of SrTiO3 NPs were well-matched with JCPDS card no. 35–0734 corresponding to cubic structured SrTiO3 nanocrystals. The XRD peaks of Cu2Se QDs were well-matched with and JCPDS card no. 29–0575 corresponding to tetragonal structured Cu2Se nanocrystals. Moreover, the corresponding XRD peaks of SrTiO3 NPs and Cu2Se QDs were all observed in the XRD spectrum of SrTiO3/Cu2Se NPs, which further demonstrated the successful synthesis of high purity SrTiO3/Cu2Se NPs. All above observations confirmed the successful fabrication of the heterojunction structure and their potential thermoelectric property of SrTiO3/Cu2Se NPs.

Fig. 1

Characterization of SrTiO/CuSe p-n heterojunction. (a) TEM images of SrTiO3 NSs (scale bar 100 nm). (b) TEM images of SrTiO3/Cu2Se NSs (scale bar 100 nm). (c) HRTEM image (inset: FFT diffraction patterns) of SrTiO3 (scale bar 5 nm). (d) HRTEM image (inset: FFT diffraction patterns) of Cu2Se (scale bar 5 nm). (e) 2D AFM image of SrTiO3/Cu2Se NSs (scale bar 100 nm for all panels). (f) 3D AFM image of SrTiO3/Cu2Se NSs. (g) phase image of the piezoelectric response of SrTiO3/Cu2Se NSs. (h) piezoelectric potential of SrTiO3/Cu2Se NSs. (i and j) SEM-EDX mapping images of SrTiO3 and SrTiO3/Cu2Se NSs (scale bar 100 nm for all panels).

Fig. 2

Chemical composition and thermoelectric characterization. (a) XPS spectra of SrTiO3 NSs, Cu2Se NPs, and SrTiO3/Cu2Se NSs. (b) XRD spectra of SrTiO3 NSs, Cu2Se NPs, and SrTiO3/Cu2Se NSs. Thermoelectric characteristics of SrTiO3 NSs and Cu2Se NPs: (c) σ, (d) S, (e) PF (f) κ, (g) ZT, and (h) S vs ln (σ), respectively.

The biomedical applications of nanomedicine largely depend on their physiological dispersibility and stability. It was observed that the surfaces of SrTiO3/Cu2Se NPs were slightly negatively charged after the hydrothermal process, (Fig. S3), which allowed surface modification by using amphipathic DSPE-PEG through hydrophobic interaction. The zeta potential of SrTiO3/Cu2Se NPs increased to −30 mV, demonstrating a successful PEG modification and thus ensuring their physiological dispensability and stability. Around 25% (w/w) of DSPE-PEG was loaded on the surface of the SrTiO3/Cu2Se NPs as measured by inductively coupled plasma-atomic emission spectrometry (ICP-AES). PEGylation of SrTiO3/Cu2Se NPs showed improved dispersion in cell culture medium, phosphate buffer saline (PBS) and water in contrast with the bare SrTiO3/Cu2Se NPs due to lack of aggregation (Fig. S4). And the size of SrTiO3/Cu2Se NPs after PEGylation increased to 153 nm and the PDI of SrTiO3/Cu2Se NPs after PEGylation decreased to 0.102. In addition, the Fourier transform infrared (FT-IR) absorption bands of the PEGylated SrTiO3/Cu2Se NPs at ∼1250 cm−1 and ∼2900 cm−1 are corresponded to the CO stretching vibration and –CH vibrations in the DSPE-PEG segment (Fig. S5). Finally, Sr (red), Ti (green), and O (blue) appeared in the energy dispersive spectrometry (EDS) mapping of SrTiO3 NPs, and after Cu2Se QDs and PEG coating, Cu (purple), Se (olive), C (yellow), and N (white) showed again in the EDS mapping of PEGylated SrTiO3/Cu2Se NPs (Fig. 1i and Fig. S6), further confirmed successful surface coating by DSPE-PEG.

Next, the thermoelectric performance of our prepared SrTiO3/Cu2Se NPs were examined. The ZT of SrTiO3 NPs and Cu2Se QDs were tested and calculated, respectively. Fig. 2c shows the material-dependent σ as a function of temperature. It is apparently that σ increases monotonically with increasing the temperature for these two samples and roughly follows a co-efficient of T−1.5, suggesting that acoustic phonons dominate the carrier scattering. Fig. 2d shows the variations of S with the temperature. The positive signal of S indicates the p-type nature for Cu2Se QDs, and the negative signal of S indicates the n-type nature for SrTiO3 NPs. S increases gently upon increasing the temperature. Fig. 2e presents the plots of S2σ as a function of temperature, from which relatively high S2σ of SrTiO3 NPs and Cu2Se QDs were obtained. Fig. 2f is the plots of κ as a function of temperature, in which the relatively low κ of SrTiO3 NPs and Cu2Se QDs were also obtained. Due to the obtained high S2σ as well as low κ, significantly enhanced ZT values are expected. Fig. 2g is the ZT plots as a function of temperature, in which a peak ZT of 0.11 and 0.17 at 333 K is achieved in the fabricated SrTiO3 NPs and Cu2Se QDs. Fig. 2h shows the S at room temperature as a function of the natural logarithm of σ of SrTiO3 NPs and Cu2Se QDs. This linear relationship between the S and the natural logarithm σ indicates more fluctuating carrier concentration and less varying carrier mobility. All above observations confirmed the good thermoelectric properties of SrTiO3/Cu2Se NPs.

SrTiO3/Cu2Se NPs mediated reactive oxygen species (ROS) generation

Followingly, the light-thermal-electricity-chemical energy conversion of SrTiO3 NPs, Cu2Se QDs, and SrTiO3/Cu2Se NPs were measured and analyzed. In order to better prediction of application effects in vivo, the initial temperature was set at 35 °C. Fig. 3a and b shows the light-thermal conversion of SrTiO3/Cu2Se NPs, in which the temperature increased from 35 °C to 45 °C under 2.5 min 808 nm laser irradiation and cooled down to 35 °C following 9 min natural cooling process. As shown in Fig. 3h, according to thermoelectric effect, under temperature gradient (35 °C–45 °C) induced by 808 nm laser irradiation and natural cooling, a build-in electric field was constructed on the opposite surfaces of SrTiO3 NPs or Cu2Se QDs. Therefore, the built-in electric field can facilitate separation of charges (electrons and holes) in bulk, and promote their transfer to the catalyst surface, making them effective tools for catalyzing reduction and oxidization of O2 and H2O to generate superoxide anion (·O2−) and hydroxyl radical (·OH), respectively. ·O2− generation through the reduction of electrons was measured with dihydrorhodamine 123 (DHR 123) probe. As shown in Fig. 3c and e, obvious fluorescence increase was detected following the temperature gradient (35 °C–45 °C) using SrTiO3 NPs and Cu2Se QDs separately, which indicated that SrTiO3 NPs and Cu2Se QDs are suitable nanomedicines for thermoelectric therapy. Of note, a much stronger fluorescence increase was observed when SrTiO3/Cu2Se NPs were applied as thermoelectric catalysts for catalyzing ·O2− generation from O2. As exhibited in Fig. 3h, after SrTiO3 NPs (n type) contacting with Cu2Se QDs (p type), an interfacial electric field was then constructed on their interface, in which the separated electrons and holes induced by thermoelectric effect further transferred and re-located on the surface of different catalysts following interfacial electric field. Thereby, the recombination of electrons and holes was restricted, leading to extending lifetime of separated electrons and holes for further redox reactions. The p-n heterojunction enhanced thermoelectric effect of SrTiO3/Cu2Se NPs was further assessed in terms of hydroxyl radical (·OH) production using methylene blue (MB) as the specific probe of ·OH. Consistent with the above results, the SrTiO3/Cu2Se NPs exhibited the strongest ·OH generation, which further confirmed their p-n heterojunction enhanced thermoelectric effects (Fig. 3d and f). By employing 5,5-dimethyl-1-pyrroline N-oxide as the spin trapping agent, electron spin resonance (ESR) was applied to detect the generated ROS directly. As shown in Fig. 3g, ·O2− and ·OH synchronously generated from SrTiO3/Cu2Se NPs through thermoelectric effects from O2 and H2O were detected, which further confirms the high ability of ROS generation of SrTiO3/Cu2Se NPs. In order to detect the potential application of SrTiO3/Cu2Se NPs in clinic, the light-thermal-electricity-chemical energy conversion of SrTiO3 NPs, Cu2Se QDs, and SrTiO3/Cu2Se NPs were also tested under irradiation of 808 nm laser at 0.33 W/cm2. As shown in Fig. S7, the ROS generation performance, including ·O2− and ·OH, were similar with that under irradiation of 808 nm laser at 1 W/cm2, which showed a huge potential for clinical application. In addition, in order to confirm the ROS generation was resulted from the thermoelectric effect, the ROS generation performances were further detected through directly heating the SrTiO3 NPs, Cu2Se QDs, and SrTiO3/Cu2Se NPs. As exhibited in Fig. S8, obvious ROS generations were observed and the yields of ROS were similar with that under irradiation of 808 nm laser at 1 or 0.33 W/cm2, demonstrating the mechanism of ROS generation was the thermoelectric effect of thermoelectric materials.

Fig. 3

ROS generation and mechanism of TET. (a) Photothermal images of SrTiO3/Cu2Se NSs under 808 nm laser irradiation. (b) photothermal conversion of SrTiO3/Cu2Se NSs under 808 nm laser irradiation. (c) and (e) ·O2− generation of SrTiO3/Cu2Se NSs under temperature gradient from 35 °C to 45 °C. (d) and (f) ·OH generation of SrTiO3/Cu2Se NSs under temperature gradient from 35 °C to 45 °C. (g) The signal of ROS generated by SrTiO3/Cu2Se NPs in EPR spectra. (h) The mechanism of SrTiO3/Cu2Se NSs mediated TET.

In vitro antitumor evaluation mediated by SrTiO3/Cu2Se NPs

The biocompatibility of the prepared TE agents was next tested using MCF 7 and Hela cancerous cells. As shown in Fig. 4a and S7, similar with the traditional photothermal agent (graphene, G), the TE agents showed negligible cytotoxicity in the absence of excitation, and more than 80% of the cells were viable even when exposed to 100 μg/mL of the respective TE agents. Stimulation with 808 nm laser led to the temperature increasing from 35 °C to 45 °C, and thus increased the cytotoxic effects of all TE agents (Fig. 4b and S8). However, under the uniform 808 nm laser irradiation and temperature increase, the cells treated with G still remain a relatively high viability. To this end, we speculated that the cytotoxic effects of TE agents are probably attributed to the thermoelectric effect inducing ROS generation, rather than photothermal effect inducing thermal. With increasing cycles of this temperature gradient (35 °C–45 °C), an enhanced cell cytotoxic effects of TE agents were observed, meanwhile, the cytotoxic effects of G remain negligible improved. This observation further demonstrated the thermoelectric effect of TE agents inducing ROS generation was the main cause for their cytotoxicity. Moreover, the cells treated with SrTiO3/Cu2Se NPs exhibited the highest cytotoxicity, confirming the p-n heterojunction enhanced thermoelectric effect. Additionally, the intracellular ROS levels under different treatments were analyzed by using fluorescent probe. Indeedly, the intracellular ROS levels were significantly higher in the SrTiO3 NPs and Cu2Se QDs treated groups, compared to those treated with G, and the highest ROS concentration was detected in cells exposed to the SrTiO3/Cu2Se NPs coupled with 808 nm laser irradiation and a natural cooling process (Fig. 4d and e). As previously reported [44], DNA damage caused by ROS is one of the main causes for ROS-induced cell toxicity. Thus, the levels of DNA damage in MCF 7 cells after different treatments were further analyzed using γ-H2AX as a marker for DNA double-strand breaks. As shown in Fig. 4f, MCF 7 cells treated with G with ΔT (35 °C–45 °C) did not show any obvious DNA damage compared with the control group. However, MCF 7 cells treated with SrTiO3 NPs or Cu2Se QDs coupling with the same ΔT, showed apparently detectable levels of DNA damage, further supporting the TE effect of SrTiO3 NPs and Cu2Se QDs. Treatment with SrTiO3/Cu2Se NPs and ΔT produced considerably high levels of irreparable DNA damage in the cancer cells. All these results thus suggest that the developed therapeutic strategy based on SrTiO3/Cu2Se NPs and ΔT with an enhanced TE effect can specifically kill cancer cells. Moreover, the efficient apoptosis of SrTiO3/Cu2Se NPs via TE effect was further detected through co-staining cells with propidium iodide (PI, dead cells, red fluorescence) and calcein AM (live cells, green fluorescence) after different treatments. The LSCM images of co-stained cancer cells, as shown in Fig. 4g, also confirmed the antitumor effect of TET in vitro.

Fig. 4

(a) Human breast cancer cells (MCF 7) viability treated with SrTiO3 NSs, Cu2Se NPs, and SrTiO3/Cu2Se NSs at different concentrations for 24 h. The data show mean ± s.d., n = 6 biologically independent cells. (b) human breast cancer cells (MCF 7) viability treated with SrTiO3 NSs, Cu2Se NPs, and SrTiO3/Cu2Se NSs and 808 nm laser at different concentrations for 24 h. The data show mean ± s.d., n = 6 biologically independent cells. (c) Antitumor effect of SrTiO3 NSs, Cu2Se NPs, and SrTiO3/Cu2Se NSs based TET under different temperature gradient cycles. The data show mean ± s.d., n = 6 biologically independent cells. (d) CLSM images of ROS content in MCF 7 cells under different treatments. Scale bar = 100 μm. (e) Fluorescence quantitative analysis of the intracellular ROS in Fig. d. (f) Representative CLSM images of the MCF 7 cells after different treatments. Scale bar = 20 μm. The nuclei were stained by DAPI (blue), and the γH2AX foci per nucleus were stained by anti-γH2AX antibody (red). (g) Live/Dead cell staining assay in MCF 7 cells after different treatments (green, live cells; red, dead cells). Scale bar = 100 μm.

In vivo antitumor evaluation mediated by SrTiO3/Cu2Se NPs

The anti-cancer potential of TET was next evaluated in vivo using MCF 7 tumor-bearing mice. The mice were each injected intravenously with Cy7-labeled NPs at the dosage of 5 mg/kg, and the fluorescence intensity of Cy7 in the blood was measured at different time intervals. As shown in Fig. 5b, the Cy7-loaded NPs remained significantly longer in circulation compared to free Cy7, which was suggestive of greater tumor accumulation of NPs. Additionally, the tumor accumulation of NPs was also confirmed by fluorescence imaging of major organs after 24 h i.v. injection (Fig. 5c). To more precisely characterize the biodistribution of NPs in vivo, an ICP was employed to test the concentration of NPs in the major organs and tumors over 24 h, which also showed a great tumor accumulation of the prepared NPs. The MCF 7 tumor-bearing mice were randomly divided into the following groups and treated accordingly: 1) saline control, 2) SrTiO3/Cu2Se NPs, 3) SrTiO3 NPs + ΔT, 4) Cu2Se QDs + ΔT, 5) SrTiO3/Cu2Se NPs + ΔT, 6) G + ΔT, and 7) PTT (G, >55 °C). The ΔT means temperature gradient (35 °C–45 °C) for 3 cycles inducing by 808 nm laser irradiation and natural cooling after 24 h post injection (Fig. 5a and e). The tumor volume was measured every 2 days, and as shown in the growth curves in Fig. 5f and g, the untreated and non-ΔT controls did not show any significant inhibition of tumor growth. The combination of TE agents (SrTiO3 NPs or Cu2Se QDs) and ΔT achieved an obvious inhibitory effect, which is attributed to the generation of ROS by the thermoelectric effect. Due to the p-n heterojunction enhanced thermoelectric effect, SrTiO3/Cu2Se NPs with ΔT markedly inhibited tumor growth, in which SrTiO3/Cu2Se NPs almost completely ablated the tumors under the same conditions. In contrast, the G NSs with the same ΔT treatment exhibited only a slight inhibition of tumor growth compared with the control group, which indicated the temperature of 45 °C can not able to induce cancer cells death. Only the hyperthermia (>55 °C) induced by G NSs-mediated PTT could provide a similar anti-tumor effect as that of TET (45 °C) (Fig. 5h). However, as shown in Fig. 5g, because the hyperthermia (>55 °C) randomly propagated and diffused to the surrounding tissues, an obvious PTT-related toxicity and side effects on normal tissues was observed, in which the skin and muscle at the 808 nm laser irradiation site were scorched. By contrast, there was negligible damage to the skin at the irradiated sites of TET (45 °C) (Fig. 5f). These findings are consistent with the thermoelectric effect and heterojunction structure of SrTiO3/Cu2Se NPs that induces an intracellular ROS burst. The representative images of mice from the different treatments are shown in Fig. 5f and g. Moreover, the mice treated with SrTiO3/Cu2Se NPs + ΔT showed the longest lifetime without any tumor recurrence (Fig. 5l). No significant changes were observed in the body weight of the mice during the experimental period (Fig. S10), indicating negligible adverse effects of this therapy in vivo.

Fig. 5

(a) Schematic illustration of treatment schedule. (b) Blood circulation performance of NPs-Cy7 and free Cy7. The data show mean ± s.d., n = 3 biologically independent animals. (c) Fluorescence images of major organs with i.v. injection of Cy 7 functionalized SrTiO3/Cu2Se NPs. (d) Biodistribution of SrTiO3/Cu2Se NPs in MCF 7 tumor-bearing mice by ICP measurement. The data show mean ± s.d., n = 3 biologically independent mice. (e) Photothermal heating and natural cooling images of tumor-bearing nude mice. (f) Anti-tumor effects of TET. Treatment 1: PBS, Treatment 2: SrTiO3/Cu2Se NPs, Treatment 3: SrTiO3 NSs + ΔT, Treatment 4: Cu2Se NPs + ΔT, and Treatment 5: SrTiO3/Cu2Se NPs + ΔT. The injected doses of SrTiO3/Cu2Se NPs, SrTiO3 NPs, and Cu2Se QDs were 6 mg/kg. (g) Traditional graphene mediated PTT. Treatment 6: graphene with temperature increase to 45 °C and Treatment 7: graphene with temperature increase to 55 °C. (h) Tumors weight after different treatments. The data show mean ± s.d., n = 5 biologically independent mice. (i) In vivo ROS detection in the sections from tumors by dihydroethidium (DHE) via fluorescence microscopy. (j) Immunofluorescence (IF) and (k) H&E staining in the sections from the tumors after different treatments. The nucleus was stained by DAPI (blue), damaged DNA was stained by γH2AX foci (red), and apoptotic cells were stained by apoptosis marker C-CAS3 (green). Scar bar = 100 μm. Three times each experiment was repeated independently with similar results. (l) The survival curves of MCF 7 tumor-bearing mice after different treatments.

The intracellular ROS burst effect was further validated by using DCFH fluorescence probe. As shown in Fig. 5i and S11, the different treatments led to consistent ROS accumulation in the tumors, and the strongest green fluorescence was detected in the SrTiO3/Cu2Se NPs + ΔT group, further indicating a drastic ROS burst in tumor cells. Given that ROS induce apoptosis through DNA damage [44], we next analyzed the tumors for signs of DNA double strand breaks, oxidative stress and apoptosis. SrTiO3/Cu2Se NPs alone induced negligible γ-H2AX foci and very few apoptotic cells, whereas both DNA damage and apoptosis were considerably higher under SrTiO3 NPs or Cu2Se QDs with ΔT. Coupling SrTiO3/Cu2Se NPs and ΔT led to a marked increase in γ-H2AX foci and apoptosis in the tumors (Fig. 5j and k). Furthermore, the levels of 8-hydroxy-2′-deoxyguanosine (8-OHdG), a marker of oxidized DNA, were consistent with that of γ-H2AX (Fig. S12). Taken together, the p-n TE agents heterojunction can efficiently trigger a ROS burst in cancer cells and induce apoptosis under a mild condition.

Biocompatibility evaluation of SrTiO3/Cu2Se NPs

In order to more precisely characterize the metabolism of SrTiO3/Cu2Se NPs in vivo, an inductively coupled plasma emission spectrometer (ICP) was employed to analyze the concentration of SrTiO3/Cu2Se NPs in major organs and tumors over 30 days. Besides the accumulations in tumors, high accumulations of SrTiO3/Cu2Se NPs were also found in other normal organs such as liver, lung, and kidneys. However, the accumulate SrTiO3/Cu2Se NPs in normal organs could be gradually excreted by the body over time (Fig. S16). Moreover, the biocompatibility of TET was evaluated via hematological, histological and immunological indices. As shown in Fig. 6a–c, no obvious difference in ROS levels, DNA damage, apoptosis, and tissue damage were observed in normal tissues after treatment with SrTiO3/Cu2Se NPs + ΔT compared with those treated with PBS, indicating a favorable biocompatibility of the SrTiO3/Cu2Se NPs and TET in normal tissues. Moreover, the serum levels of IFN-γ, IL-6, TNF-α and IL-12+P40 were similar in the control and treated mice 12 and 24h post i.v. injection of SrTiO3/Cu2Se NPs (10 mg/kg) (Fig. 6d). In addition, routine blood examination on days 1, 7 and 14 post-injection did not show any significant differences in aspartate aminotransferase (AST), alanine aminotransferase (ALT), white blood cells (WBC), blood urea nitrogen (BUN), alkaline phosphatase (ALP), red blood cells (RBC), platelet (PLT), Hemoglobin (HGB), mean corpuscular volume (MCV), creatinine (Cr), lymphocyte (LYM), hematocrit (HCT), and neutrophil (NEU) between the control and treated groups (Fig. 6e). Taken together, SrTiO3/Cu2Se NPs and TET are biocompatible in vivo.

Fig. 6

/CuSe NPs and SrTiO/CuSe NPs mediated TET. H&E staining and immunofluorescence (IF) staining in the sections from the major organs, which were collected from the MCF 7 xenograft-tumor bearing mice after different treatments with (a) PBS, (b) SrTiO3/Cu2Se NPs, and (c) SrTiO3/Cu2Se NPs mediated TET. The nucleus was stained by DAPI (blue), damaged DNA was stained by γH2AX foci (red), and apoptotic cells were stained by apoptosis marker C-CAS3 (green). Scar bar = 100 μm. Three times each experiment was repeated independently with similar results. (d) Serum levels IFN-γ, IL-6, TNF-α, and IL-12+P40 in healthy mice at 2 or 24 h post intravenous injection of PBS or SrTiO3/Cu2Se NPs. e Blood biochemistry and hematology analysis of Balb/c mice treated with PBS or SrTiO3/Cu2Se NPs.

Comparation of side effects between PTT and TET

As demonstrated above, PTT with hyperthermia (>55 °C) could easily damage the skin at irradiated site. To further confirm the superiority of TET, we finally compared the treatment-related toxicity and side effects on normal tissues and organs through simulating the PTT and TET at some major organs and tissues. The PTT (55 °C) and TET (45 °C) related toxicity to major organs and tissues were simulated by directly exposing these organs and tissues to 55 °C and 45 °C. As exhibited in Fig. 7, exposure to the TET (45 °C) conditions, negligible toxicity or side effect are observed in their H&E staining images of heart, liver, spleen, lung, kidney, muscle, and skin, compared with these without any treatment. However, obvious and serious damages were revealed in these important organs and tissues after exposing to the PTT (55 °C) conditions. For example, congestion, enlargement of intercellular space, tissue defect, etc., were presented in these major organs. Additionally, evident swelling and critical damage were also observed in muscle and skin under treated with PTT (55 °C). All above phenomena further confirmed the in vivo safety of TET and demonstrated competitive advantages over PTT.

Fig. 7

H&E staining of major organs and tissues contacted with different temperature stimulating the potential damages of TET and traditional PTT to nearby organs and tissues. Scar bars = 500 μm.

Conclusions

Cancer therapies based on energy conversion, such as photothermal therapy (PTT, light-to-thermal energy conversion) and photodynamic therapy (PDT, light-to-chemical energy conversion) have attracted extensive attention in preclinical research. However, the PTT-related hyperthermia (>55 °C) damage to surrounding tissues and shallow penetration of PDT-applied light (visible region) prevent further advanced clinical practices. The thermoelectric therapy (TET) based on NIR-to- tepidity-chemical energy (ROS) conversion, not only effectively avoids the defects of PTT and PDT, but also integrates the advantages of PTT and PDT. Here, a novel TET based on p-n heterojunction TE generator was successfully developed and demonstrated outstanding anticancer potency with negligible side-effects. The SrTiO3/Cu2Se NPs based p-n heterojunction was prepared by simple two-step hydrothermal processes, exhibiting an excellent thermoelectric effect under mild temperature gradient from 35 °C to 45 °C. The formation of build-in electric field induced by thermoelectric effect under temperature gradient allowed directional separation of electrons and holes in the bulk of SrTiO3 NPs and Cu2Se QDs. Furthermore, the interfacial electric field induced by contacting of SrTiO3 NPs (n type) and Cu2Se QDs (p type) further guided the distribution and re-location of the excited electrons and holes onto the surface of SrTiO3 NPs and Cu2Se QDs, respectively. The synergy between build-in and interfacial electric fields facilitated the electrons and holes separation and transfer both in the bulk and the interface, minimizing the undesired charge recombination. Under 808 nm laser irradiation and natural cooling induced temperature gradient (35–45 °C), the engineered SrTiO3/Cu2Se NPs serve as an intelligent TE generator with dually independent ROS (·O2− and ·OH) generation through catalyzing the oxidation and reduction of O2 and H2O in tumor microenvironment. With an effective ROS burst mediated apoptosis of cancer cells both in vitro and in vivo, the p-n heterojunction TE generator based TET has been demonstrated to be a novel and potential clinic cancer treatment. This work is also expected to provide a smart strategy for the design of other p-n heterojunction TE generator with efficient charges separation and will inspire future studies in expanding their in-depth application, especially in other biomedical applications, such as diabetic ulcer treatment and wound infection resistance under temperature difference between the body and outside environment. In addition, with the vigorous development of tumor immunotherapy [[45], [46], [47], [48]], the combination of thermoelectric therapy and immunotherapy can more effectively eliminate tumor in situ and effectively inhibit tumor recurrence and metastasis.

CRediT authorship contribution statement

Yong Kang: Methodology, Formal analysis, Writing – original draft. Na Kong: Software, Investigation, Writing – original draft. Meitong Ou: Validation. Ying Wang: Formal analysis. Qicai Xiao: Writing – review & editing. Lin Mei: Resources. Bing Liu: Resources. Liqun Chen: Conceptualization, Supervision. Xiaobin Zeng: Conceptualization, Writing – review & editing, Supervision, Funding acquisition. Xiaoyuan Ji: Conceptualization, Writing – review & editing, Supervision, Project administration, Funding acquisition.

Declaration of competing interest

The authors declare no conflict of interest.