Fabrication and characterization of RS-Te
The sodium tellurite (Na2TeO3) was added into the medium during the culture of Sal, and rod-like nanostructures were observed inside Sal by TEM (Fig. 1A). Meanwhile, the color of Sal suspension turned to black (Additional file 1: Fig. S1), which may be attributed to the reduction of Te oxyanions via the intracellular glutathione (GSH), reduced nicotinamide dinucleotide phosphate and GSH reductase in Sal and the spontaneous decomposition of the obtained Te precursor (GSTeH) [16]. A large number of Sal could be found in the RAW264.7 cell by TEM after incubating the S-Te (Fig. 1B), and the TeNPs in the Sal could also be clearly observed through magnified TEM (Fig. 1C). The elements composition and distribution of RS-Te were analyzed by elemental mapping images, containing Te and elements from Sal (P, S, and N) (Fig. 1D). After the macrophages and Sal with green fluorescent protein (GFP) were co-cultured, bright green fluorescence was observed in a large number of RAW264.7 cells, suggesting successful uptake of the Sal by the RAW264.7 cells (Additional file 1: Fig. S2). To further characterize the bacterial uptake behavior of macrophages, the RAW264.7 cell membrane was labeled with a red fluorescent dye (DiI), and the nucleus was located via 4',6-diamidino-2-phenylindole (DAPI, blue fluorescence). The GFP was visually observed in the RAW264.7 cells by CLSM (Fig. 1E), indicating that Sal was successfully taken up by RAW264.7 cells. The XRD pattern of Te from RS-Te matched well with the standard diffraction pattern (JCPDS No. 36-1452) of hexagonal Te, in which the diffraction peaks of Te could be indexed to (100), (101), (110), (201), and (113) (Fig. 1F). The valence state of Te from RS-Te was analyzed by X-ray photoelectron spectroscopy. As shown in Fig. 1G, the characteristic peaks at binding energies of 572.9 and 583.0 eV were assigned to Te 3d5/2 and Te 3d3/2 of Te0, while the characteristic peaks at binding energies of 575.5 and 585.9 eV were assigned to Te 3d5/2 and Te 3d3/2 of tellurium oxide, which might be attributed to the easily-occurred tellurium oxide in natural environment [17]. Compared to the size of a single RAW264.7 cell, there was no significant change observed in the size of RS-Te (Additional file 1: Fig. S3A), and the surface charges of both the single RAW264.7 cell and RS-Te were found to be comparable (Fig. 3B). These findings suggest that RAW264.7 cells maintained their integrity after internalizing Sal-containing Te. In addition, SDS-PAGE (Fig. 1H) analysis showed that RS-Te had similar protein bands compared with the profile of S-Te and single RAW264.7 cell, indicating that the surface proteins of the Sal were still present after the Sal were ingested by RAW264.7 cells. The result is helpful for the activation of the immune response in vivo.
Studies have shown that tellurates and tellurites are toxic, and they could inhibit bacterial growth [18]. As demonstrated in Additional file 1: Fig. S4A, with the increase of Na2TeO3 concentration, the absorption value (OD600) of Sal gradually decreased, and a concentration of 0.8 mM had a significant inhibitory effect on Sal growth. When the above samples were further diluted (1×104) and cultured on the solid LB plates, that the Sal multiplication ability was significantly inhibited compared with the untreated group (Additional file 1: Fig. S4B). These results showed that the mechanism of cytoplasmic synthesis of TeNPs in Sal might be related to the detoxification of tellurates and tellurites, by generating Te0 and tellurium oxide with less toxicity. To balance high biomineralization efficiency and low proliferation rate, the 0.8 mM Na2TeO3 was chosen for the preparation of the S-Te.
Photothermal properties of RS-Te
Rod-like TeNPs based on polypeptide-mineralization have shown to have excellent photothermal conversion and tumor ablation capabilities in our previous work [19], and it was reported that PTT elicited immunogenic cell death (ICD) by inducing dying tumor cells to release damage-associated molecules [20]. The UV-vis-NIR absorption spectra of Te showed that Te presented a wide light absorption spectrum, which is positively correlated with the concentration of Te (Fig. 2A). To explore whether the biomineralized S-Te has acceptable photothermal properties, a thermal imager was introduced to record the temperature changes of the different concentrations of S-Te under NIR laser irradiation (808 nm, 1.5 W/cm2). As shown in Fig. 2B, Te concentrations in S-Te suspensions are positively correlated with temperature increases. The corresponding quantitative data are shown in Fig. 2C. Upon irradiation with an NIR laser, the temperature of S-Te suspension at Te concentration of 40 µg/mL increased by 35.8°C, while water only slightly increased by 4.3°C. Under NIR laser irradiation for 10 min, the temperature changes of S-Te (40 µg/mL) at different power densities (0.75, 1.0, and 1.5 W/cm2) were measured to be 16.7, 23.4, and 33.8°C, respectively, suggesting a power density-dependent photothermal effect of S-Te (Fig. 2D). These obvious temperature changes provided favorable conditions for in vivo applications of S-Te. In addition, S-Te exhibited a similar temperature rise as TeNPs from Sal at the same condition, while dead Sal and water showed negligible temperature changes. The results reveal the photothermal property from TeNPs rather than bacteria in photothermal heating of S-Te (Fig. 2E). It is worth noting that there was little difference between the temperature changes of the RS-Te and S-Te upon NIR laser irradiation (Te: 40 µg/mL, 808 nm, 1.5 W/cm2), indicating that the double-camouflaged strategy had little effect on the photothermal properties of the delivery platform (Fig. 2F). It was found that the temperature curve was an inappreciable change after 5 repeated irradiation cycles, revealing the remarkable photothermal conversion stability of RS-Te (Fig. 2G). Moreover, the time constant (τs) was measured to be 219.36 s, and photothermal conversion efficiency was calculated to be 33.8% (η), which is higher than that of common photothermal nanoagents in the PTT (Fig. 2H, I), such as Au nanorods (22%), CuS nanoparticles (28.8%), and Pd nanosheets (30.9%) [21–23]. Together, these results indicate the superior NIR photothermal performance and high photostability of RS-Te, implying its potential for PTT of tumors.
In vitro immune stimulation performance induced by RS-Te-triggered PTT
As one of the key immune cells involved in cancer immunity, macrophages are one of the most abundant circulating cells in the body [24]. To investigate whether biomineralized S-Te could polarize macrophages into M1 macrophage cells, FCM was introduced to analyze the ratio of M1 and M2 in macrophage samples after different treatments. As shown in Fig. 3A, an significant increase of the macrophages M1/M2 rations was found in raw macrophages incubated with S-Te, RS-Te, dead Sal, and LPS (the positive control), while the update in the TeNPs group was negligent comparing with the untreated group, which implies that the Sal played a key role in the polarization of macrophages. Furthermore, the proportional ratio increase of M1/M2 macrophages in the RS-Te group might be caused by secreting chemokines by RS-Te this chemokines induce macrophage polarization. To further investigate the effect of RS-Te on TAMs remodeling, IL-4-conditioned RAW264.7 cells (M2 macrophages) were incubated with PBS, TeNPs, RS-Te, S-Te, and LPS for 12 h (Fig. 3B). The proportion of M1/M2 was significantly increased in the S-Te, RS-Te, dead Sal, and LPS groups compared with the TeNPs and PBS groups. Correspondingly, the mean fluorescence intensity (MFI) of CD86+ (M1 macrophage marker) significantly increased in S-Te, RS-Te, dead Sal, and Sal groups compared with PBS and TeNPs groups (Fig. 3C). Besides, the secretion of proinflammatory factors, such as TNF-α and IL-12, markedly increased in IL-4-conditioned RAW264.7 cells (Additional file 1: Fig. S5A, B). The contents of M2 macrophage-related cytokines in the samples were also detected by FCM, such as TGF-β and IL-10. Moreover, their levels were found to be significantly decreased in the S-Te, RS-Te, dead Sal, and LPS groups (Additional file 1: Fig. S5C, D), suggesting an effective polarization from the M2 phenotype to the M1 phenotype.
Antigen-presenting cells (APCs) play a key role in the initiation and regulation of innate and adaptive immune responses. Once exposed to antigens, the immature DCs transform into maturation for antigen processing and presenting [25]. Therefore, DCs maturation induced by RS-Te was analyzed by FCM to measure the expression levels of co-stimulatory molecules (CD80, CD86, and MHC-II). Compared with the PBS group, the expression levels of CD80+ CD86+ in BMDCs incubated with dead Sal and S-Te increased 2.52 and 2.47 times, respectively. However, no obvious change was found when BMDCs incubated with TeNPs, indicating that it was Sal in S-Te that promoted the maturation of DCs in vitro (Fig. 3D). It is worth noting that the expression levels of CD80+ CD86+ in the RS-Te group were 1.67 times higher than that of the S-Te group, which might be due to the fact that RS-Te enriched with more Sal and that the polarized RS-Te further matured the BMDCs by secreting related cytokines. The expression levels of MHC-II also presented a similar trend in BMDCs with different treatments (Fig. 3E). To investigate whether RS-Te-trigged PTT maturate DCs in vitro, supernatants and 4T1 cell debris after various treatments were collected and incubated with BMDCs for 12 h. As shown in Fig. 3F, G, compared with the control group, supernatants and 4T1 cell debris incubated with either S-Te or RS-Te could induce higher expression of CD80+ CD86+ and MHC-II of BMDCs, whether plus or not. These results indicate that the bacteria and M1 macrophage could act as immune adjuvant for DC maturation induction. For all groups with laser irradiation, DC maturation could be further promoted. In particular, BMDCs in 4T1 + RS-Te + Laser group exhibited the highest expression of CD80+ CD86+ and MHC-II among all groups, indicating that ICD induced by photothermal was another major player for DCs maturation. Moreover, the transwell models were established to simulate RS-Te-triggered PTT in TME. 4T1 cells were cultured in the upper chamber and BMDCs were cultured in the bottom chamber (Fig. 3H). Then, the upper chamber was separately treated with PBS, LPS, RS-Te, and RS-Te + Laser. The FCM assay results show that RS-Te, and RS-Te + Laser treated BMDCs significantly increased the expression of costimulatory molecules, including CD86 and MHC-II (Additional file 1: Fig. S6A, B). Correspondingly, the secretion of proinflammatory factors, such as TNF-α and IL-6, also increased (Additional file 1: Fig. S6C, D). In particular, after NIR laser irradiation, the levels of TNF-α and IL-6 in the RS-Te + Laser group were 2.18 and 2.33 times higher than that of the RS-Te group, respectively. Considering the importance of the role of MHC molecules in antigen presentation and impressive ICD induced by RS-Te, RS-Te-triggered PTT has a great advantage in synergistically enhancing tumor immunotherapy. To further explore whether RS-Te could stimulate DCs maturation in vivo, the inguinal lymph nodes from healthy mice are collected after 24 h subcutaneous injection of RS-Te. The percentages of mature DCs in inguinal lymph nodes showed a significant elevation compared with the control group (Fig. 4A, B), which confirms the ability of RS-Te to stimulate DCs maturation in vivo. All of the results imply that RS-Te could transform M2 macrophages into M1 macrophages, and has the potential to remodel the immunosuppressive TME and promote antitumor immune responses.
Antitumor effects and immune responses of RS-Te-mediated photothermal immunotherapy in vitro
As shown in Fig. 3D, E, RAW264.7 remained active after incubating the S-Te. The live/dead cells were respectively stained by calcein-AM (green fluorescence) and PI (red fluorescence) to evaluate the viability of RAW264.7 cells, untead RAW264.7 cells as a control (RAW), macrophages were incubated with Sal, S-Te, and LPS as RS, RS-Te, and RL groups, respectively. As shown in Fig. 4C, no red fluorescence was observed after different treatments, confirm the specificity of RS-Te. After incubating the above supernatant with 4T1 cells, the results of the CCK-8 experiment showed that the cell viability decreased to a certain extent in RS, RS-Te, and RL groups compared with the PBS and RAW groups, suggesting RS-Te secretion had tumor cells killing effect (Fig. 4D). Follow more, RS-Te was examined in vitro. As shown in Fig. 4E, 4T1 cell viabilities decreased to 73.8% after incubation with RS-Te for 24 h, compared with the 4T1 cells incubated S-Te. The results show that RS-Te could retain its normal activity and kill cancer cells, which is consistent with the previous results (Fig. 3D, E). 4T1 cells exhibited high viabilities after incubation with TeNPs, Dead + Sal, Dead + Sal + laser, and S-Te, while 4T1 cell viabilities significantly decreased at TeNRs plus laser, S-Te plus laser, and RS-Te plus laser groups for 24 h (Te concentration: 30 µg/mL). The results indicate that it was the photothermal conversion ability of TeNPs that caused the temperature hike, led to cell death. Especially, compared with the S-Te + laser group, the 4T1 cell viability decreased to 8.03% after 4T1 cells were incubated with RS-Te plus laser for 24 h. These results show the prominent photothermal therapeutic efficacy of RS-Te under NIR irradiation.
In vivo tumor targeting and biodistribution of RS-Te
To validate the tumor-homing ability of RS-Te, subcutaneous 4T1 tumor-bearing mice models were established, and the NIR fluorescence dye Cy5.5-labeled RS-Te was employed for the real-time fluorescence monitoring of in vivo biodistribution and metabolism for a long time. The corresponding results are illustrated in Fig. 5A. After intravenous injection of the RS-Te (Te concentration: 1.0 mg/kg), the fluorescence signal of the tumor site gradually increased, and reached to peak at 24 h, showing the excellent tumor retention capacity of RS-Te. The fluorescence signal quantization results of the tumor site in Fig. 5B are consistent with those observed above. Blood samples were then collected at different time points, and Te concentrations were measured to determine how the RS-Te metabolized in vivo over time. As shown in Fig. 5C, less RS-Te accumulated in the heart, which avoided the risk of acute toxicity by Te [26]. Especially, a fairly high concentration of Te was detected at the tumor site, verifying that RS-Te administered systemically could efficiently accumulate and retain in tumors. These data verified that the prepared RS-Te possessed high tumor-homing ability. Thanks to gratifying enrichment ability, the in vivo photothermal properties of RS-Te were verified by NIR laser irradiation at the tumor site after 24 h intravenous injection of RS-Te and PBS. The thermal imaging data (Fig. 5D) revealed that a significant temperature increase of the tumor site could be observed after NIR laser irradiation for 10 min (808 nm, 1.5 W/cm2). The temperature changes in the tumor site are recorded (Fig. 5E), the temperature of the tumor site rose rapidly within 4 min and reached 59.3°C after 10 min irradiation with NIR laser, indicating RS-Te possessed excellent photothermal conversion properties in vivo.
Enhanced biosafety of RS-Te
To assess the biocompatibility and biosafety of RS-Te, the physiological and biochemical changes were monitored in healthy mice by intravenously injecting RS-Te at a Te concentration of 1.0 mg/kg. As demonstrated in Additional file 1: Fig. S7A, administration of S-Te, RS-Te, and RS-Te + Laser induced slight weight loss and the weight loss was gradually recovery in 14 days, while all mice receiving the Sal all died within 48 h. This is possibly due to the rapid proliferation of Sal in the body. The body temperature of the mice treated with S-Te, RS-Te, and RS-Te + Laser slightly dropped and back to normal within 4 h, while the Sal-treated mice showed a significant decrease in body temperature (Additional file 1: Fig. S7B). Moreover, blood biochemistry and routine examinations were carried out at 0.5, 1, and 14 days after intravenous injection. The mice in the Sal group had died after 1 day, so the data were no longer recorded (Fig. 6). Following the injection of S-Te, there was a sharp increase of liver function marker ALT and AST and kidney function marker BUN and CRE in the serum levels on the 1st day (Additional file 1: Fig. S9B-E). Also, a spike of CRP concentration was detected on the 1st day after treatment with S-Te, manifesting the occurrence of inflammation or infection (Fig. 6F). Compared to the PBS group, these markers were slightly elevated in the RS-Te group and gradually backed to normal within 14 days, this could be an indicator that the macrophage modification strategy greatly reduced the risk of bacterial infection and significantly improved the biosafety of the delivery vector. The trend of blood biochemistry and routine indexes change in RS-Te + Laser were similar to that of the RS-Te group, indicating that laser treatment did not cause serious body damage. Overall, the system of S-Te delivery by RS-Te significantly enhanced the safety and potential for in vivo applications.
Antitumor effects and immune responses of RS-Te-mediated photothermal immunotherapy in vivo
Spurred by the prominent photothermal conversion and immune response feature of RS-Te in vitro, the in vivo antitumor effects of intravenously injected RS-Te upon NIR irradiation were evaluated. 4T1 tumor-bearing mice (5 weeks, female) were randomly divided into 4 groups (4 mice/per group). At 24 h post systemic administration of PBS or RS-Te via tail vein, the mice were illuminated with or without NIR laser irradiation (1.5 W/cm2, 10 min), and the 4 groups were denoted as PBS, PBS + Laser (PBS + L), RS-Te, and RS-Te + Laser (RS-Te + L) groups, respectively. Tumor growth was barely inhibited in the PBS and PBS + L groups (Fig. 7A). Although tumor growth was slower in the RS-Te group compared with the PBS and PBS + L groups, the tumor volume reached to 659.87 cm3 during the observation period. On the contrary, after NIR laser treatment, tumors in the RS-Te + L group were almost eliminated. The tumor growth status is shown in Fig. 7B. On day 24, all mice were euthanized, and the tumors were collected and photographed (Fig. 7C). The mean tumor weight in each group was calculated (Fig. 7D). The tumor volume and weight were comparable with monitoring data. Moreover, the H&E, Ki67, and TUNEL staining showed that the RS-Te + L group initialized the most potent apoptosis effect and the lowest proliferation of tumor cells (Fig. 7E). Those results indicated that the RS-Te had excellent PTT ability. The other groups showed a slight body weight loss within two days of different treatments except for the PBS group. This may be explained by mice's excessive stress response, and the body weight of mice gradually recovered and gained a slight amount of weight (Fig. 7F). At the end of treatment, the major organs of mice were sectioned and analyzed (Fig. 7G). The tissue structure of the PBS, RS-Te, and RS-Te + L groups was similar to that of the PBS group, and no obvious structural damage was observed, which confirms the safety of the RS-Te. Since tumor nodules were found in the PBS group but not in the RS-Te + L group, we further carred out H&E staining for the lungs tissue of each group (Additional file 1: Fig. S8). Compared with the large number of tumor nodules in the lungs in the PBS group, the tumor nodules were significantly smaller after intravenous injection of RS-Te and NIR laser irradiation, indicated that RS-Te plus laser treatment possessed strong anti-tumor metastasis potential.
To elucidate the mechanism of tumor elimination and anti-tumor metastasis triggered by RS-Te-based phototherapy, 4T1 tumor-bearing mice (five weeks, female) were randomly divided into 4 groups (4 mice/per group). The mice were intravenously injected with PBS, S-Te, and RS-Te, after 24 h post injection. Another group of RS-Te was treated with laser irradiation (1.5 W/cm2, 10 min), denoted as PBS, S-Te, RS-Te, and RS-Te + L groups, respectively. DLNs and tumors of mice were collected and analyzed by FCM at 48 h after administration. It is shown that intravenous administration of RS-Te induced the more pronounced proliferation of DCs maturation in DLNs compared with administration of S-Te, indicating that the delivery system of macrophages carrying mineralized bacteria had a stronger immune activation capacity than that of bacterial vectors alone (Additional file 1: Fig. S9). More importantly, RS-Te plus laser treatment led to further DC maturation, and a similar trend of mature DC proportion was observed in tumors of each group, illustrating that the combination of Te-based photothermal killing and biomimetic strategy could synergistically contribute to DC maturation (Additional file 1: Fig. S10). The population of tumor-infiltrating effector T lymphocytes (CD3+ CD4+ T cells and CD3+ CD8+ T cells) in total T cells and Tregs in the tumor tissues after treatments were measured. As shown in Fig. 7H, although the level of CD3+ CD8+T cells in S-Te group was elevated relative to the PBS group, the level of CD3+ CD8+T cells increase was more significant in RS-Te and RS-Te + L groups, where the level of CD3+CD8+T cells in RS-Te + L group was 4.84, 2.54, and 1.32 times higher than that of the PBS, S-Te, and RS-Te groups, respectively. A similar change trend of CD3+CD4+T cells in tumor tissues was also found (Fig. 7I). In contrast, Tregs that play an important role in immune suppression showed a 5.64-fold reduction relative to the PBS group (Fig. 7J). Moreover, the level of TAMs in tumors was confirmed by measuring the protein markers from macrophage cell surfaces, including CD80+ CD86+ and CD80+ CD206+. As shown in Fig. 7K, L, RS-Te treating boosted the percentage of CD80+ CD86+ with a rate of 17.03%, which was 12.05% in the PBS group and 14.69% in the S-Te group. It is also observed that RS-Te plus NIR laser treatment caused the highest proportion (22.98%) of CD80+ CD86+, which might be Sal, such as by carrying shRNA against a critical gene of metastasis or upregulating tumor genes, expressing effector proteins, such as IFN-γ [27]. The IFN-γ was subsequently examined, and the level of IFN-γ obviously increased in the RS-Te + laser group (Fig. 7M). In addition, the serum levels of TNF-α and IL-6 in the RS-Te + laser group were also remarkably aggrandized (Fig. 7N, O), which are consistent with the in vitro results. All these results verify that RS-Te could efficiently reprogram the immunosuppressive TME and induce strong CD8+ and CD4+ T cells immune response, attributing to the significantly augmented macrophage polarization, in situ tumor-associated antigens (TAAs) release and presentation, and pro-inflammatory factor regulation, and these responses were further reinforced under NIR laser irradiation.