Ultrasmall PEI-Decorated Bi2Se3 Nanodots as a Multifunctional Theranostic Nanoplatform for in vivo CT Imaging-Guided Cancer Photothermal Therapy

Bi-based nanomaterials, such as Bi2Se3, play an important part in biomedicine, such as photothermal therapy (PTT) and computed tomography (CT) imaging. Polyethylenimine (PEI)-modified ultrasmall Bi2Se3 nanodots were prepared using an ultrafast synthetic method at room temperature (25°C). Bi2Se3 nanodots exhibited superior CT imaging performance, and could be used as effective photothermal reagents owing to their broad absorption in the ultraviolet–visible–near infrared region. Under irradiation at 808 nm, PEI-Bi2Se3 nanodots exhibited excellent photothermal-conversion efficiency of up to 41.3%. Good biocompatibility and significant tumor-ablation capabilities were demonstrated in vitro and in vivo. These results revealed that PEI-Bi2Se3 nanodots are safe and a good nanotheranostic platform for CT imaging-guided PTT of cancer.


INTRODUCTION
The early diagnosis of cancer and "precision" medicine are major challenges for oncologists. Scientists need to develop multifunctional biocompatible nanotheranostic platforms that integrate diagnostic and therapeutic functions (Barreto et al., 2011;Ho et al., 2015;Chen et al., 2016). As a non-invasive method, phototherapy can alleviate the side-effects and suffering of treatment as compared with that using resection, chemotherapy, or radiotherapy Fan et al., 2017;Lei et al., 2018;Basak et al., 2019).
We report a facile room-temperature method for synthesizing ultrasmall polyethylenimine-decorated Bi 2 Se 3 (PEI-Bi 2 Se 3 ) nanodots for CT imaging-guided PTT of cancer in vitro and in vivo. Compared with strategies reported previously, the rapid synthesis of PEI-Bi 2 Se 3 nanodots may improve the efficiency of the synthesis and prevent further surface modification. Furthermore, the raw materials are inexpensive, and the organic solvents are nontoxic and environmentally friendly, thereby making the process suitable for future production at a large scale.
The synthesized nanodots exhibited excellent absorption properties and the efficiency of photothermal conversion was high under laser irradiation at 808 nm. Moreover, the outstanding CT imaging and photothermal-ablation capacity observed in vitro and in vivo, and non-significant long-term toxicity observed in vivo, revealed that PEI-Bi 2 Se 3 nanodots could achieve CT imaging-guided PTT of cancer. Hence, PEI-Bi2Se3 nanodots could be powerful and safe nanotheranostic agents in cancer therapy.

RESULTS AND DISCUSSION
The Synthesis and Characterization of PEI-Bi 2 Se 3 Nanodots A novel NIR light-responsive nanotheranostic platform based on PEI-Bi 2 Se 3 nanodots for CT imaging-guided PTT of cancer was fabricated (Scheme 1). Under the protection of an inertgas atmosphere and ice-water bath, Se powder was reduced by NaBH 4 under magnetic stirring at room temperature (25°C) to obtain NaHSe solution (Se 2− precursor solution). An appropriate amount of the Se 2− precursor solution was SCHEME 1 | Fabrication of PEI-Bi 2 Se 3 nanodots for CT imaging-guided PTT of cancer.
Frontiers in Pharmacology | www.frontiersin.org December 2021 | Volume 12 | Article 795012  Frontiers in Pharmacology | www.frontiersin.org December 2021 | Volume 12 | Article 795012 3 transferred rapidly to a mixed solution of ethylene glycol and water containing Bi(NO 3 ) 3 ·5H 2 O and PEI. The reaction solution turned black rapidly, indicating that the reaction was ultra-facile and efficient (within 1 min). In the Experimental section in Supplementary Material, the experimental details are presented. Moreover, Bi 2 Se 3 nanodots had promising potential for CT imaging-guided PTT of cancer owing to an efficient photothermal performance, strong absorption in the NIR region, and the high X-ray attenuation coefficient of Bi 3+ .
A transmission electron microscopy (TEM) image of the obtained PEI-Bi 2 Se 3 nanodots is shown in Figure 1A. A clear lattice fringe with a distance of 0.304 nm can be seen on the highresolution TEM image ( Figure 1B), which can be attributed to the (015) planes of Bi 2 Se 3. Furthermore, the prepared PEI-Bi 2 Se 3 nanodots, as uniform spheres with relatively narrow size distribution, had a mean diameter of 3.56 nm ( Figure 1C). The X-ray diffraction (XRD) patterns of the prepared PEI-Bi 2 Se 3 nanodots are shown in Supplementary Figure S1. All the characteristic XRD peaks matched well with the standard hexagonal phase of Bi 2 Se 3 (Joint Committee on Powder Diffraction Standards 33-0214). Moreover, PEI-Bi 2 Se 3 nanodots could maintain good dispersity in various solutions, such as Dulbecco's modified Eagle's medium, phosphate-buffered saline (PBS), NaCl, and water, for several months (Supplementary Figure S2), indicating that the PEI modification was beneficial for improving the stability of PEI-Bi 2 Se 3 nanodots.
Photothermal Performance of PEI-Bi 2 Se 3 Nanodots in vitro PEI-Bi 2 Se 3 nanodots showed a broad ultraviolet-visible-NIR (UV-Vis-NIR) absorption spectrum ranging from 500 to 1,100 nm ( Figure 2A). As the concentration of Bi 3+ increased, the absorption intensity of PEI-Bi 2 Se 3 nanodots was enhanced, and the colorless solution turned dark black (Supplementary Figure S3). Absorbance at 808 nm increased linearly (Supplementary Figure  S4), which suggested that PEI-Bi 2 Se 3 nanodots exhibited good dispersibility in water, and could be excellent photothermal agents for PTT. To investigate the photothermal performance, pure water (control) and aqueous solutions of PEI-Bi 2 Se 3 nanodots (25, 50, 100, 200 μg/ml) were exposed to a NIR laser (808 nm, 1.0 W cm −2 ) for 10 min. The temperatures of the different PEI-Bi 2 Se 3 nanodot solutions increased rapidly, exhibiting noticeable concentrationand irradiation time-dependent behavior ( Figure 2B). This finding indicated that the temperature of the PEI-Bi 2 Se 3 solution could reach up to 70.1°C at a concentration of 200 μg ml −1 (1.0 W cm −2 , 10 min), which is highly effective for killing tumor cells via hyperthermia. In contrast, under identical experimental conditions, the temperature of pure water increased by up to 8.4°C ( Figure 2C). The IR thermal images of pure water and aqueous solution of PEI-Bi 2 Se 3 nanodots (200 μg ml −1 ) after a certain duration of irradiation are shown in Figure 2D. The temperature of the PEI-Bi 2 Se 3 aqueous solution (200 μg ml −1 ) increased rapidly with an increase in the irradiation duration using a laser at 808 nm whereas, under identical conditions, the temperature of pure water increased slowly. PEI-Bi 2 Se 3 nanodots could therefore convert NIR energy into thermal energy rapidly and efficiently, and act as potential photothermal agents during PTT. In particular, the efficiency of photothermal conversion of PEI-Bi 2 Se 3 nanodots was up to 41.3% ( Figures 2E,F), which is much higher than that of currently reported photothermal agents, such as PVP-Bi nanodots (∼30%) (Lei et al., 2017), Bi 2 Se 3 nanosheets (∼33%) (Xie et al., 2017), and Cu 2−x Se nanocrystals (∼22%) (Hessel et al., 2011). Photostability is another prerequisite for evaluating the performance of photothermal agents during PTT. After irradiation of the aqueous solution of PEI-Bi 2 Se 3 nanodots (200 μg ml −1 ) using a continuous-wave NIR laser at 808 nm for 1 h (1.0 W cm −2 ), the color of the solution, UV-Vis-NIR spectrum, and morphology exhibited no distinct changes ( Supplementary  Figures S5, S6). Hence, PEI-Bi 2 Se 3 nanodots possessed satisfactory photothermal stability. All the results shown above (excellent photothermal effect and good photostability) highlighted the potential of PEI-Bi 2 Se 3 nanodots as suitable agents for PTT of cancer.

Studies on Cytotoxicity and Photothermal Ablation of Tumor Cells
Evaluation of the cytotoxicity of PEI-Bi 2 Se 3 nanodots is important. The cytotoxicity of PEI-Bi 2 Se 3 nanodots was tested by the Cell Counting Kit-8 assay. Even at a high concentration of PEI-Bi 2 Se 3 nanodots (200 μg ml −1 ), the viability of A549 cells was 96 and 92% after incubation for 24 h (red bars) and 48 h (green bars), respectively ( Figure 3A). These results indicated that PEI-Bi 2 Se 3 nanodots exhibited no distinct toxicity towards A549 cells.
Because of the outstanding photothermal performance of PEI-Bi 2 Se 3 nanodots, we investigated their photothermal effects against tumor cells. The IR thermal images of A549 cells incubated with PEI-Bi 2 Se 3 nanodots in a 96-well plate are shown in Figure 3B. Notably, the temperature could increase up to 53.8°C under irradiation at 808 nm (1.0 W cm −2 ) after addition of PEI-Bi 2 Se 3 nanodots to the culture. Cancer cells are sensitive to heat, and can be killed effectively at >42°C. To identify further the anti-cancer effect of PEI-Bi 2 Se 3 nanodots on A549 cells, live and dead cells were imaged using a fluorescence microscope after staining with calcein acetoxymethyl ester (green fluorescence) and propidium iodide (red fluorescence), respectively. In the control groups, notable cytotoxicity was not observed (PBS, PEI-Bi 2 Se 3 nanodots only, laser only), whereas almost no living cells were observed in the PEI-Bi 2 Se 3 + laser group ( Figure 3C). These results suggested that the as-synthesized PEI-Bi 2 Se 3 nanodots with low cytotoxicity would produce satisfactory results upon in vivo cancer treatment. In addition, endocytosis pathways were determined in order to identify the uptake mechanism of extracellular PEI-Bi 2 Se 3 nanodots. The cellular uptake of PEI-Bi 2 Se 3 was evaluated by monitoring the fluorescence of FITC in the A549 cells at various incubation times (1, 3, and 6 h). As shown in Supplementary Figure S7, green fluorescence of FITC was observed after 1 h incubation with FITC-labeled PEI-Bi 2 Se 3 . The

CT Imaging in vitro and in vivo
High resolution, easy manipulation, and high penetrability make CT imaging an important part of medical diagnoses (Lee et al., 2013;Du et al., 2020). Bi element with its large atomic number and high electron density has promising capacity for X-ray attenuation (Kinsella et al., 2011;Li et al., 2016). The X-ray absorption coefficient and iobitridol were compared to evaluate the in vitro CT imaging capability of PEI-Bi 2 Se 3 nanodots. The latter exhibited a much higher CT density than that of iobitridol at equivalent concentrations ( Figure 4A), whereas the Hounsfield unit (HU) values of both contrast agents exhibited a typical linear dependence on the concentration ( Figure 4B). Compared with the curve for iobitridol, the curve for PEI-Bi 2 Se 3 nanodots had a steeper slope. Hence, the as-synthesized PEI-Bi 2 Se 3 nanodots had superior ability in CT imaging and were effective contrast agents. Inspired by the satisfactory CT effect in vitro, we assessed the feasibility of using PEI-Bi 2 Se 3 nanodots as CT contrast agents in vivo. Time-dependent CT imaging was undertaken after tumorbearing mice were injected (i.v.) with PEI-Bi 2 Se 3 nanodots (Bi concentration 30 mM, 150 μL). At 0 h-1 h after injection, the CT density at the tumor site brightened gradually ( Figure 4C), which was caused by passive accumulation of PEI-Bi 2 Se 3 nanodots at the tumor site through the enhanced permeability and retention effect. Thereafter, the density decreased because some PEI-Bi 2 Se 3 nanodots had metabolized. The mean HU value   Figure 4D). These results demonstrated that PEI-Bi 2 Se 3 nanodots could serve as promising in vivo CT contrast agents for the accurate diagnosis of cancer.
Photothermal Effect, Photothermal Therapy, and Long-Term Toxicity of PEI-Bi 2 Se 3 Nanodots in vivo Based on the good properties of PEI-Bi 2 Se 3 nanodots at tumor sites (i.e., excellent in vitro photothermal effect, satisfactory CT imaging effect, and outstanding passive targeted accumulation), we studied the feasibility of CT imaging-guided PTT of cancer in vivo. PEI-Bi 2 Se 3 nanodots (Bi concentration 20 mg kg −1 ) were injected (i.v.) into tumor-bearing mice. Obvious enhancement was observed 1 h after injection, so the tumor was irradiated using a laser at 808 nm (1.0 W cm −2 ) 1-h later, and the photothermal effect in vivo was monitored by an IR thermal camera. The temperature at the tumor site in the treatment group increased rapidly as the duration of irradiation increased ( Figure 5A) but the temperature at the tumor site did not show a significant change compared with that in the control group. The corresponding curve detailing temperature variation is shown as Figure 5B. After 10 min of NIR irradiation (808 nm, 1.0 W cm −2 ), in the presence of PEI-Bi 2 Se 3 nanodots, the temperature at the tumor sites was as high as 51.1°C whereas, in the control group, a minor increase in temperature was observed. Hence, PEI-Bi 2 Se 3 nanodots could serve as excellent photothermal agents for in vivo tumor ablation: they could kill cancer cells and inhibit their continued diffusion.
A tumor model was established by injecting (s.c.) U14 cells into the left axilla of female Kunming mice. Once the tumors had grown to ∼100 mm 3 , mice were used for experimentation. The mice bearing the U14 cells were divided randomly into four groups of six: 1) control; 2) laser only; 3) PEI-Bi 2 Se 3 nanodots; 4) PEI-Bi 2 Se 3 nanodots + laser. The tumors in mice were irradiated (808 nm) 1 h after injection of PEI-Bi 2 Se 3 nanodots. The bodyweight and tumor volume of mice were measured every 2 days to evaluate therapeutic efficacy. After various treatments, the bodyweight of mice showed a steady increase ( Figure 5C), thereby indicating that PEI-Bi 2 Se 3 nanodots did not produce toxic side-effects during PTT. The tumor volume of each mouse was measured using a Vernier caliper, and plotted as a function of the relative tumor volume and treatment duration ( Figure 5D). The average weights of excised tumors are shown in Figure 5E, and representative tumor photographs of each group are shown in Figure 5F. Compared with groups 1-3, the growth of tumors in group 4 was inhibited significantly after 14 days of PTT. In addition, hematoxylin and eosin (H&E) staining revealed no appreciable damage in groups 1-3; simultaneously, severe shrinkage and discrete cancer cells were observed clearly in group 4 ( Figure 5G). Taken together, these results demonstrated that PEI-Bi 2 Se 3 nanodots possessed potential as ideal and safe photothermal agents for cancer treatment.
The potential long-term toxicity of PEI-Bi 2 Se 3 nanodots in vivo was also investigated. Thirty days after injection, pathological samples of major organs (heart, lungs, liver, spleen, kidneys) from control mice and treated mice were obtained. H&E staining ( Figure 6) revealed no distinct tissue damage or inflammatory lesions in any major organ. Moreover, there were no abnormal signs in treated mice during the entire observation period. These results confirmed that PEI-Bi 2 Se 3 nanodots were not significantly toxic in vivo.

CONCLUSION
Ultrasmall PEI-Bi 2 Se 3 nanodots were fabricated via an ultrafast, facile, and environmentally friendly method. The obtained PEI-Bi 2 Se 3 nanodots could ensure good contrast enhancement owing to their high X-ray attenuation coefficient, and showed good photothermal killing effects in vitro and in vivo owing to considerable photothermal-conversion effects. Moreover, PEI-Bi 2 Se 3 nanodots possessed negligible long-term toxicity in vivo. Therefore, we believe that the as-synthesized PEI-Bi 2 Se 3 nanodots are useful theranostic agents for CT-imaging-guided PTT of cancer.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.

ETHICS STATEMENT
The animal study was reviewed and approved by the Laboratory Animal Center of Jilin University.