p-n Heterojunction-based Thermoelectric Generator for Highly Efficient Cancer Thermoelectric Therapy


 Photothermal therapy (PTT) based on the light-heat conversion principle has attracted extensive attention in preclinical research, however, the hyperthermia resulted the treatment-related damage to surrounding tissues prevents further advanced clinical practice. Here, we developed a thermoelectric therapy (TET) based on p-n heterojunction (SrTiO3/Cu2Se nanoplates) on the principle of light-heat-electricity-chemical energy conversion, demonstrating great potential for cancer treatment. The principle of TET is based on light-heat-electricity-chemical energy conversion, regarded as an upgraded version of PTT. Upon laser irradiation and subsequently natural cooling-induced the mild temperature gradient (35-45 oC), a self-build-in electric field was constructed and thereby facilated electrons and holes separation in bulk SrTiO3 and Cu2Se. Importantly, the contact between SrTiO3 (n type) and Cu2Se (p type) constructed another interficial electric field, which further guided the seperated electrons and holes to transfer to re-locate onto the surfaces of SrTiO3 and Cu2Se, respectively. The formation of two electric fields in bulk and interface of SrTiO3/Cu2Se nanoplates minimized probability of charges recombination. Of note, high-performance superoxide radicals (·O2−) and hydroxyl radicals (·OH) generation from O2 and H2O under catalization by seperated electrons and holes, led to intracellular ROS burst and cancer cells apoptosis without apparent damage to surrounding tissues. As far as it is known, this is the first report on TET based a p-n heterojunction in biomedical field. Construction of bulk and interficial electric fields in heterojunction for improving charges separation and transfer is also expected to provide a robust and universal strategy for diverse applications including energy, environment, and biomedical engineering.


Introduction
Photothermal therapy (PTT), which is based on light-heat conversion, has aroused comprehensive attention and emerged as a potential cancer treatment strategy due to its spatiotemporal addressability, minimal invasiveness, and short treatment time. [1][2][3][4] To achieve a high anticancer e ciency, two essential criteria of traditional PTT should be considered, including high photo-thermal conversion performance of the employed photothermal agent (PTA) and long light penetration with minimized tissue scattering and absorption. 5,6 Typically, photothermal conversion e ciency is the critical factor for an eligible PTA, and it is quite necessary for a PTA to increase the temperature of the tumor site above 50 o C for achieving a desired therapeutic outcome. Spurred by recent progress in nanotechnology, various PTAs, including gold nanostructure, 7-10 carbon nanomaterials (graphene and its analogs), [11][12][13][14][15][16][17] and the recently developed twodimensional nanomaterials (transition metal dichalcogenides (TMDCs), carbides and nitrides (MXenes), and monoelemental materials (Xenes)) [18][19][20][21][22][23][24] , have currently emerged as e cient materials for PTTmediated cancer treatment. 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 light. [25][26][27][28][29] Nevertheless, only limited examples of NIR-II PTAs 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. [30][31][32][33] Therefore, despite these great advances achieved to date, most of the existing PTTs are not speci cally related to cancer-associated events. That is, although light irradiation could target the tumor site, hyperthermia induced by conventional PTAs or ambient uid 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 heat to electricity via electron-hole pairs separation under temperature gradient-induced the build-in thermoelectric eld, have attracted tremendous attention worldwide in materials science and solid-state physics, due to their wide application in Peltier cooling and waste energy harvesting. [34][35][36][37][38][39][40] Further analysis of the mechanism of TE materials shows the separated electron-hole pairs under build-in thermoelectric eld demonstrating great potentials in catalyzing reactive oxygen species (ROS) generation in cushy conditions, similar with the mechanisms of photodynamic therapy and piezocatalytic therapy. 41,42 In addition, compared with PTTgenerated 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 elds of energy and environment, it is still an infant in biomedical elds. Typically, the thermoelectric gure of merit is the key to evaluate the e ciency of TE materials, ZT = S 2 sT/k, where S, s, T and k represents Seebeck coe cient, electrical conductivity, temperature, and thermal conductivity, respectively. 43,44 Obviously, the thermoelectric e ciency not only relates to physicochemical properties of TE materials, such as Seebeck coe cient, 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 e ciency, which is also the main obstacle for applications in biomedical elds of TE materials. 45 Based on our previous studies, 46-50 heterojunction construction, including p-n junction and Z scheme junction, has been demonstrated as an e cient strategy for improving electron-hole pairs separation and enhancing the catalytic e ciency. After contacting of p-type and n-type semiconductors, the interfacial electric eld will be constructed, in which the separated electrons and holes in both semiconductors would transfer in the opposite direction and locate in different semiconductors. [51][52][53][54] The unfavorable recombination of electron-hole pairs would be retarded, which is the key for conversion e ciency of photocatalysts, electrocatalysts, and TE materials.
Herein, for the rst time, we developed a novel thermoelectric therapy (TET) based on light-heat-electricitychemical energy conversion, by employing SrTiO 3 (n type) and Cu 2 Se (p type) to construct a p-n heterojunction, which is capable of dual independently targeted generating ROS under mild temperature gradient (from 35 o C to 45 o C). As shown in Figure 1, by employing conventional two-step hydrothermal processes, SrTiO 3 /Cu 2 Se based p-n heterojunction was constructed. Under 808 nm laser irradiation and natural cooling, the electrons and holes in the bulk of SrTiO 3 and Cu 2 Se voluntarily separate and migrate from the bulk to the surface under the driving force of the build-in thermoelectric eld on the opposite directions. Additionally, the p-n heterojunction between SrTiO 3 and Cu 2 Se constructs an interfacial electric eld, and thus redistributes the surface electrons and holes to speci c 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 e cient 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.

Results And Discussion
Preparation and characterization of SrTiO 3 /Cu 2 Se p-n heterojunction In the rst stage of this work, the SrTiO 3 /Cu 2 Se p-n heterojunction was synthesized through two steps of hydrothermal process. Detailly, as illustrated in Figure 1, SrTiO 3 nanoplates (NPs) (n type TE materials) were papered rstly following Cu 2 Se quantum dots (QDs) synthesis and in site coating on the surface of  Figure   2h) obtained by PFM in the darkness, which demonstrates its piezoelectric property and further testi es its thermoelectric property. To further con rm the successful fabrication and the composition of SrTiO 3 NPs, Cu 2 Se QDs and SrTiO 3 /Cu 2 Se NPs, X-ray photoelectron spectroscopy (XPS) and X-ray diffractometry (XRD) were performed. In the XPS analysis (Figure 3a), the speci c 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 SrTiO 3 /Cu 2 Se NPs. In the XRD spectrum (Figure 3b), two respective structures corresponding with SrTiO 3 and Cu 2 Se were observed. All above observations con rmed the successful fabrication of the heterojunction structure and their potential thermoelectric property of SrTiO 3 /Cu 2 Se NPs.
The biomedical applications of nanomedicine largely depend on their physiological dispersibility and stability. It was observed that the surfaces of SrTiO 3 /Cu 2 Se NPs were slightly negatively charged after the hydrothermal process, ( Figure S3), which allowed surface modi cation by using amphipathic DSPE-PEG through hydrophobic interaction. The zeta potential of SrTiO 3 /Cu 2 Se NPs increased to -30 mV, demonstrating a successful PEG modi cation and thus ensuring their physiological dispensability and stability. Around 25% (w/w) of DSPE-PEG was loaded on the surface of the SrTiO 3 /Cu 2 Se NPs as measured by inductively coupled plasma-atomic emission spectrometry (ICP-AES). PEGylation of SrTiO 3 /Cu 2 Se NPs showed improved dispersion in cell culture medium, phosphate buffer saline (PBS) and water in contrast with the bare SrTiO 3 /Cu 2 Se NPs due to lack of aggregation ( Figure S4). In addition, Next, the thermoelectric performance of our prepared SrTiO 3 /Cu 2 Se NPs were examined. The ZT of SrTiO 3 NPs and Cu 2 Se QDs were tested and calculated, respectively. Figure 3c shows the materialdependent s as a function of temperature. It is apparently that s increases monotonically with increasing the temperature for these two samples and roughly follows a co-e cient of T -1.5 , suggesting that acoustic phonons dominate the carrier scattering. Figure 3d shows the variations of S with the temperature. The positive signal of S indicates the p-type nature for Cu 2 Se QDs, and the negative signal of S indicates the n-type nature for SrTiO 3 NPs. S increases gently upon increasing the temperature. NPs and Cu 2 Se QDs were obtained. Figure 3f is the plots of κ as a function of temperature, in which the relatively low κ of SrTiO 3 NPs and Cu 2 Se QDs were also obtained. Due to the obtained high S 2 s as well as low κ, signi cantly enhanced ZT values are expected. Figure 3f 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 SrTiO 3 NPs and Cu 2 Se QDs. Figure 3h shows the S at room temperature as a function of the natural logarithm of s of SrTiO 3 NPs and Cu 2 Se QDs. This linear relationship between the S and the natural logarithm s indicates more uctuating carrier concentration and less varying carrier mobility. All above observations con rmed the good thermoelectric properties of SrTiO 3 /Cu 2 Se NPs. NPs exhibited the strongest ·OH generation, which further con rmed their p-n heterojunction enhanced thermoelectric effects (Figure 4d and 4f). By employing 5,5-dimethyl-1-pyrroline N-oxide as the spin trapping agent, electron spin resonance (ESR) was applyed to detect the gnerated ROS directly. As shown in Figure 4g, ·O 2 − and ·OH synchronously generated from SrTiO 3 /Cu 2 Se NPs through thermoelectric effects from O 2 and H 2 O were detected, which further con rms the high ability of ROS generation of SrTiO 3 /Cu 2 Se NPs.
In vitro antitumor evaluation mediated by SrTiO 3 /Cu 2 Se NPs The biocompatibility of the prepared TE agents was next tested using MCF 7 and Hela cancerous cells.
As shown in Figure 5a 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 mg/mL of the respective TE agents. Stimulation with 808 nm laser led to the temperature increasing from 35 o C to 45 o C, and thus increased the cytotoxic effects of all TE agents (Figure 5b 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 heat. With increasing cycles of this temperature gradient (35 o C -45 o C), an enhanced cell cytotoxic effects of TE agents were observed, meanwhile, the cytotoxic effects of G remain negligiblely 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 SrTiO 3 /Cu 2 Se NPs exhibited the highest cytotoxicity, con rming the p-n heterojunction enhanced thermoelectric effect. Additionaly, the intracellular ROS levels under different treatments were analyzed by using uorescent probe. Indeedly, the intracellular ROS levels were sign cantly higher in the SrTiO 3 NPs and Cu 2 Se QDs treated groups, compared to those treated with G, and the highest ROS concentration was detected in cells exposed to the SrTiO 3 /Cu 2 Se NPs coupled with 808 nm laser irradiation and a natural cooling process (Figures 5d and 5e). As previously reported, 58 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

In vivo antitumor evaluation mediated by SrTiO 3 /Cu 2 Se 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 uorescence intensity of Cy7 in the blood was measured at different time intervals. As shown in Figure 6b, the Cy7loaded NPs remained sign cantly longer in circulation compared to free Cy7, which was suggestive of greater tumor accumulation of NPs. Additionally, the tumor accumulation of NPs was also con rmed by uorescence imaging of major organs after 24 h i.v. injection (Figure 6c). 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 hours, 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) SrTiO 3 /Cu 2 Se NPs, 3) SrTiO 3 NPs + DT, 4) Cu 2 Se QDs + DT, 5) SrTiO 3 /Cu 2 Se NPs + DT, 6) G + DT, and 7) PTT (G, >55 o C). The DT means temperature gradient (35 o C -45 o C) for 3 cycles inducing by 808 nm laser irradiation and natural cooling after 24 h post injection (Figure 6a and 6e). The tumor volume was measured every 2 days, and as shown in the growth curves in Figure 6f  The intracellular ROS burst effect was further validated by using DCFH uorescence probe. As shown in Figure 6i and S11, the different treatments led to consistent ROS accumulation in the tumors, and the strongest green uorescence was detected in the SrTiO 3 /Cu 2 Se NPs + DT group, further indicating a drastic ROS burst in tumor cells. Given that ROS induce apoptosis through DNA damage, 58

Comparation of side effects between PTT and TET
As demonstrated above, PTT with hyperthermia (>55 o C) could easily damage the skin at irradiated site. To further con rm the superiority of TET, we nally 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. As exhibited in Figure 8, exposure to the TET (45 o 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 o 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 o C). All above phenomena further con rmed the in vivo safety of TET and demonstrated competitive advantages over PTT.

Conclusions
In summary, a novel TET based on p-n heterojunction TE generator was successfully developed and demonstrated outstanding anticancer potency with negligible side-effects. The SrTiO 3 /Cu 2 Se NPs based p-n heterojunction was prepared by simple two-step hydrothermal processes, exhibiting an excellent 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 e cient 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.

Synthesis of SrTiO 3 /Cu 2 Se NPs
The SrTiO 3 /Cu 2 Se NPs based p-n heterojunction was prepared by simple two-step hydrothermal processes. First, SrTiO 3 NPs were synthesized by a hydrothermal method using Te on lined stainless steel autoclave containing the mixture of (CH 2 OH) 2 , NaOH, Sr(NO 3 ) 2 , and Ti(OBu) 4  Next, the DCFH-DA solution ( nal concentration of 0.2 μM) was put into the above medium and incubated for 0.5 hours. 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/cm 2 and cooled naturally for 10 min. The green uorescence 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 hours (37 °C, 5% CO 2 ). Then the SrTiO 3 NPs, Cu 2 Se QDs or SrTiO 3 /Cu 2 Se 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 hours. Following culture medium removing and PBS washing, the cells were illuminated for 2.5 min employing an 808 nm light with power 1 W/cm 2 and cooled naturally for 10 min. After 24-hour 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 SrTiO 3 /Cu 2 Se NPs (5 mg/kg) were i.v. injected in C57BL/6 mice. After different intervals, a microplate reader was utilized to test uorescent intensity of Cy7 through the collected 20 μL blood.

In vivo imaging and biodistribution study
For the sale of uorescence imaging and biodistribution study in vivo, 200 μL of Cy7 functionalized SrTiO 3 /Cu 2 Se NPs (5 mg/kg) were i.v. injected into mice bearing MCF 7 tumors. The Maestro2 In-Vivo Imaging System was employed to detect the uorescent intensity of tumor and major organs 24 hours post-injection.
In vivo toxicity C57BL/6 mice were i.v. injected with SrTiO 3 /Cu 2 Se NPs (10 mg/kg) in order for toxicity analysis in vivo.

Statistical analysis
Data statistics and statistical signi cance 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.