Preparation And Characterization Of Micelles
In accordance with our previous reports, AIE-1 was synthesized and characterized [42]. The micelles AIE-M and AIE/Biotin-M were prepared through a simple ultrasonic treatment in the absence or presence of a tumor-targeting polymer (DSPE-PEG2000-Biotin). As depicted in Fig. 1A, the size distribution and average hydrodynamic diameter of AIE-M and AIE/Biotin-M, measured by DLS, were 19.2 nm and 20.5 nm with a narrow distribution (PDI = 0.098 and 0.103), respectively. Moreover, Fig. 1B showed that after being stored at 4°C for a week, both AIE-M and AIE/Biotin-M remained stable, with only minor changes in size distribution. The morphologies and diameter sizes of AIE-M and AIE/Biotin-M were observed in a dried state and calculated with TEM and image J. As shown in Fig. 1C and D, AIE-M and AIE/Biotin-M in a dried state were spherical in shape with 18.3 and 20.2 nm of average diameters, respectively. It was demonstrated that compared with AIE-M, AIE/Biotin-M endowed with tumor-targeted element exhibited negligible changes in diameter size and shape. These results showed that AIE/Biotin-M was suitable for cancer therapy. Our previous work has demonstrated that AIE-1 has no fluorescence emission when dissolved in a good solvent, while exhibiting strong fluorescence emission in an aqueous solution. As depicted in Fig. 1E, the photophysical properties of AIE-1/DPPC-PEG2000-Biotin in DMSO indicated that without fluorescence emission was detected at the excitation of 365 nm. However, the fluorescence intensity of AIE-1/DPPC-PEG2000-Biotin in DMSO was gradually enhanced with the increase of water fraction, which is a typical characteristic of AIEgens.
Therefore, DPPC-PEG2000-Biotin in AIE/Biotin-M has negligible influence on the photophysical properties of AIE-M.
Our previous work has demonstrated that chelating groups in AIE-M could form a stable complex with Cu2+, which impaired the fluorescence emission of this probe [42]. In addition to chelation with Cu2+, we further found that AIE-M and AIE/Biotin-M could chelate with Fe3+ and Zn2+. The fluorescence emission of AIE-M and AIE/Biotin-M gradually changed with the addition of Fe3+ and Zn2+, which indicated the formation of metal ion complexes with salen groups in AIE-M and AIE/Biotin-M. Notably, the fluorescence intensity of AIE-M-Zn and AIE/Biotin-M-Zn aqueous solution were gradually enhanced and reached a plateau with the addition of equivalent Zn2+. And after addition of Fe3+, the fluorescence intensity of AIE-M-Fe and AIE/Biotin-M-Fe aqueous solution gradually decreased and without completely quenched after the addition of equivalent Fe3+ (Additional file 1: Figure S1). Meanwhile, metal ions (Fe3+, Cu2+, Zn2+) have negligible influence on the average size of AIE-M and AIE/Biotin-M, Fig. 1F. These results revealed that AIE micelles could efficiently chelate with multiple metal ions (Fe3+, Cu2+, Zn2+) and form stable complexes, exhibiting potentials for multiple metal ions interference cancer therapy.
The Sonosensitivity Of Micelles
To explore the sonodynamic performance of AIE/Biotin-M, the ultrasonic stability of micelles was firstly detected. AIE-M and AIE/Biotin-M have negligible change in fluorescence emission intensity and average hydrodynamic size after separately irradiated with US (1 MHz, 2 Wcm− 2, 50% duty cycle) for 5 and 10 min (Fig. 2A-D), suggesting significant sono-stability of both AIE micelles. In the process of SDT, sonosensitizers irradiated with US can generate a large amount of ROS, such as 1O2 and ·OH. Here, we utilized 2, 2, dimethylpyrroline-1-oxide (DMPO) to separately differentiate 1O2 and ·OH in AIE micelles mediated SDT through electron spin resonance (ESR) technique. TEMP can efficiently capture 1O2 to yield 2, 2, 6, 6-tetramethylpiperidine-1-oxyl (TEMPO) and exhibit characteristic ESR signal of 1:1:1 triplet peak under static magnetic field. And DMPO can efficiently capture ·OH to yield DMPO-·OH and display a typical ESR signal of 1:2:2:1 under a static magnetic field. As depicted in Fig. 2E, both AIE micelles with US (1
MHz, 2 Wcm− 2, 50% duty cycle) irradiation for 5 min exhibited a strong triplet peak of TEMPO indicated the generation of 1O2. AIE-M and AIE/Biotin-M without US irradiation showed weak triplet peaks of TEMPO. And AIE-M and AIE/Biotin-M are equal in the triplet peak intensity of TEMPO after irradiated with US, which demonstrated tumor targeting Biotin-polymers have no effect on the sonosensitivity of AIE/Biotin-M. Furthermore, there was no characteristic ESR signal of DMPO-·OH was detected when AIE-M and AIE/Biotin-M treated with or without US irradiation (Fig. 2F), indicating no production of ·OH.
To further qualitatively and quantitatively analyze ROS generation in AIE micelles mediated SDT, SOSG and TA were exploited to detect 1O2 and ·OH, respectively. SOSG with no fluorescence emission can be oxidized by 1O2 to form SOSG-endoperoxide (SOSG-EN) and show strong fluorescence emission at 525 nm; TA with no fluorescence emission can be oxidized by ·OH to form 2-hydroxyl terephthalic acid (TAOH) and show strong fluorescence emission at 435 nm. According to fluorescence emission (Fig. 2G-I) and Additional file 1: Figure S2, US (1 MHz, 2 Wcm− 2, 50% duty cycle) treated AIE-M and AIE/Biotin-M exhibited highly characteristic SOSG-EN fluorescence signal, while no TAOH fluorescence signal could be observed. Meanwhile, the efficient generation of 1O2 positively corresponded to irradiation time, which was indicated by the SOSG-EN fluorescence signal increased over irradiation time. The ESR and fluorescence signal results demonstrated that AIE/Biotin-M could act as a highly efficient sonosensitizer to mediate sonodynamic cancer therapy by generating 1O2 under US irradiation in vitro and in vivo.
Cellular uptake and cytotoxicity
The tumor targeting ability of AIE/Biotin-M, which plays an important role in anti-tumor efficiency, was estimated towards 4T1 cells upon determination of intracellular AIEgens green fluorescence by confocal laser scanning microscope (CLSM). AIE-M and AIE/Biotin-M were separately treated with 4T1 cells for 8 h and 24 h. The green fluorescence of AIEgens in AIE-M and AIE/Biotin-M treated 4T1 cells were increased with coincubation time, Fig. 3A-C. Notably, the green fluorescence of AIEgens in AIE/Biotin-M treated 4T1 cells was higher than that in AIE-M treated cells. Quantitative analysis also showed that the green fluorescence of AIE micelles in AIE/Biotin-M treated 4T1 cells was 2.4-fold higher than that in AIE-M treated 4T1 cells after coincubation for 24 h. These results demonstrated that AIE/Biotin-M could effectively internalized by 4T1 cells with the high biotin receptor expression on cell membrane.
Theoretically, high intracellular concentration of AIEgens sonosensitizers has positive effect on the US-mediated ROS generation. To qualitatively measure the generation of ROS in cells, DCFH-DA was selected as an intracellular ROS detection probe. DCFH, which enzymatically hydrolyzed from DCFH-DA in 4T1 cells, was oxidized by ROS to form DCF with green fluorescence emission. 4T1 cells pre-treated with AIE-M and AIE/Biotin-M exhibited negligible DCF green fluorescence in the absence of US irradiation (Fig. 3D-F). After US (1.0 MHz, 2 Wcm− 2, 50% duty cycle) irradiation, both 4T1 cells pre-treated with AIE-M and AIE/Biotin-M exhibited DCF green fluorescence. And the DCF green fluorescence was increased with irradiation time, indicating intracellular generation of ROS was time-dependent in AIE micelles mediated SDT. Due to the high concentration of AIEgens in 4T1 cells pre-treated with AIE/Biotin-M, the DCF green fluorescence was stronger in AIE/Biotin-M treated cells than that in cells treated with AIE-M. Quantitative analysis demonstrated that after US irradiation, the average DCF green fluorescence intensity in 4T1 cells pre-treated with AIE/Biotin-M sharply increased 1.5-fold than that in 4T1 cells pre-treated with AIE-M. The results indicated that AIE/Biotin-M might show powerful performance on sonodynamic cancer therapy in vitro and in vivo.
Due to AIE/Biotin-M showing excellent performance on cellular internalization and 1O2 generation, it can be speculated that AIE/Biotin-M have a significant effect on anti-tumor in vitro. The cytotoxicity of AIE/Biotin-M against 4T1 cells with or without US irradiation was evaluated by standard MTT assay. As shown in Fig. 3G, even at a high concentration of 200 µg·mL-1, neither AIE-M nor AIE/Biotin-M showed toxicity against 4T1 cells in the absence of US stimulation. Similarly, merely US irradiation has no toxicity to 4T1 cells. However, under US (1.0 MHz, 2 Wcm-2, 50% duty cycle) irradiation for 3 min, both AIE-M and AIE/Biotin-M significantly inhibited the growth of 4T1 cells in a concentration-dependent manner. Differently, the sonotoxicity of AIE/Biotin-M (IC50 = 63.1 µg·mL-1) against 4T1 cells was significantly higher than that of AIE-M (IC50 = 110.5 µg·mL-1), due to the excellent cellular endocytosis of AIE/Biotin-M. The results were consistent with the intracellular generation of ROS under US irradiation.
The in vitro ICD effects
Next, we investigated whether AIE/Biotin-M treated 4T1 cells could release DMAPs in the presence of US, such as CART and HMGB1, which are a crucial characteristic of ICD. CART, calcium-binding proteins in the endoplasmic reticulum (ER), exposed on the cell outer membrane act as an “eat me” signal, stimulating
DCs and macrophages to recognize and engulf the dying tumor cells and tumor debris. As shown in CLSM measurement (Additional file 1: Figure S3A), after irradiation with US (1.0 MHz, 2 Wcm− 2, 50% duty cycle) for 3 min, AIE-M and AIE/Biotin-M showed high CATR red fluorescence. Due to the effective intracellular endocytosis of AIE/Biotin-M, CATR red fluorescence in AIE/Biotin-M pre-treated 4T1 cells was higher than the 4T1 cells pre-treated with AIE-M. Other groups, such as only US, AIE-M, and AIE/Biotin-M without US irradiation exhibited negligible CATR red fluorescence intensity. HMGB1, a nuclear protein, could release from nuclear to cytoplasm and extracellular environment, stimulating inflammatory response and inducing DCs maturation. As depicted in Additional file 1: Figure S3B, upon US irradiation (1.0 MHz, 2 Wcm− 2, 50% duty cycle) for 3 min, 4T1 cells pre-treated with AIE/Biotin-M also induced a large amount of HMGB1 transferring from nuclei to cytoplasm, exhibiting higher red fluorescence intensity in cytoplasm than other groups. Upon US irradiation, the strong expression of CART and HMGB1 in AIE/Biotin-M treated cells may due to cancer cells damage and death induced by a pronounced amount of ROS generation. These results confirmed that AIE/Biotin-M mediated SDT could efficiently cause immunogenic cancer cell death in vitro and exhibited potential in stimulating a systemically immune response in vivo.
Intratumor Accumulation Of Micelles
To optimize the experimental parameters for cancer SDT in vivo, this study applied a fluorescence imaging assay to track the in vivo behavior of AIE/Biotin-M. Due to the absorbance wavelength of AIE micelles below 600 nm, suffering from poor tissue penetration, we prepared Ce6-loaded AIE micelles to monitor the tumor accumulation of AIE-M and AIE/Biotin-M through in vivo real-time fluorescence imaging assay. The Ce6-loaded AIE/Ce6-M and AIE/Ce6/Biotin-M were prepared using the emulsification method. The average size of AIE/Ce6-M and AIE/Ce6/Biotin-M were quite consistent with that of AIE-M and AIE/Biotin-M (Fig. 4A). Therefore, Ce6-loaded micelles could be applied to explore the tumor accumulation of AIE-M and AIE/Biotin-M based on the fluorescence of Ce6. 4T1 tumor-bearing BALB/c mice were separately i.v. injected with free Ce6, AIE/Ce6/Biotin-M and AIE/Ce6-M (Ce6 2.5
mg/kg). At each time point (0–8 h), the in vivo fluorescence images were acquired after i.v. injection (Fig. 4B). Not surprisingly, free Ce6 exhibited low accumulation in the tumor, showing weak fluorescence intensity at each time point (0–8 h) after i.v. administration. While AIE/Ce6/Biotin-M and AIE/Ce6-M showed relatively strong fluorescence intensity in tumor tissues at 2 h post-injection due to enhanced permeability and retention effect (EPR). Notably, Fig. 4C quantitative analysis illuminated that AIE/Ce6/Biotin-M exhibited the highest fluorescence intensity in the tumor site and long-circulation time due to the high expression of the biotin receptor on the surface of 4T1 cells. As we all known, higher concentration of AIEgens would result in strong efficiency of SDT. Therefore, it was possible for AIE/Biotin-M to show an excellent SDT effect in vivo.
We have demonstrated that the AIE micelles could chelate with the metal ions (Cu2+, Fe3+, Zn2+) to enhance (Zn2+) or impair (Cu2+, Fe3+) the fluorescence emission of AIEgens. Therefore, we next explored the fluorescence change of AIE micelles in tumor tissue through excising tumor tissues at each time point (1–4 h) after i.v. injection of AIE-M and AIE/Biotin-M at AIE micelles dose of 100 mg·kg− 1. The excised tumor tissues were sliced and observed by CLSM. As depicted in Fig. 4D-E, although the metal-ions (Cu2+, Fe3+, Zn2+) were abundant in tumor tissues, there were detectable AIE micelles green fluorescence in tumor biopsies after i.v. injection for 1, 2, 3, 4 h. And at 2 h post-injection, AIE-M and AIE/Biotin-M exhibited high green fluorescence in tumor biopsies. Significantly, AIE/Biotin-M showed stronger green fluorescence than AIE-M in tumor biopsies at each time point (1–4 h) after i.v. injection, which was consistent with in vivo fluorescence imaging. As we illustrated that the high AIEgens green fluorescence in cells could contribute to significant SDT efficiency. Therefore, metal ions (Cu2+, Fe3+, Zn2+) in tumor tissues had negligible influence on AIE/Biotin-M mediated SDT efficiency when AIE micelles at i.v. injection dose of 100 mg·kg− 1. These results demonstrated that AIE/Biotin-M could not only chelate with multiple metal ions (Cu2+, Fe3+, Zn2+) but also show excellent potential to mediate cancer SDT in vivo.
The in vivo ICD effect and immune response
Encouraged by in vitro results, we further investigated whether AIE/Biotin-M could induce ICD and immune response upon US irradiation in vivo. 4T1 tumour-bearing mice with tumour volume 80 mm3 were randomly divided into six groups including PBS, PBS + US, AIE-M, AIE/Biotin-M, AIE-M + US and AIE/Biotin-M + US. Mice were intravenously injected with AIE-M and AIE/Biotin-M at AIE micelles dose of 100 mg/kg, once every 3 days for three times, respectively. Due to the high tumor accumulation of AIE-M and AIE/Biotin-M at 2 h post-injection, at this time point, tumors were treated with US (1 MHz, 2 Wcm− 2, 50% duty cycle) for 5 min or without US. After the last irradiation for 6 h, tumors were excised, sliced and stained for immunofluorescence imaging of CART and HMGB1. As depicted in immunofluorescence imaging (Additional file 1: Figure S4), 4T1 tumors treated with AIE-M and AIE/Biotin-M plus US irradiation showed strong CART exposure on the cell outer membrane. In line with in vitro, 4T1 tumors treated with AIE/Biotin-M plus US irradiation induced the largest amount of CART exposure. Notably, without US irradiation, 4T1 tumors treated with AIE-M and AIE/Biotin-M slightly activated CART exposure may due to AIE micelles mediated metal ions (Cu2+, Fe3+, Zn2+) depletion. Metal ions depletion in tumor tissues can reduce tumor angiogenesis, resulting in reducing/blocking the replenishment of nutrients for inhibition of tumor cells growth and metastasis. Consistent with CART, without US irradiation, 4T1 tumors treated with AIE-M and AIE/Biotin-M also slightly induced the release of HMGB1, which was demonstrated by the high signal of HMGB1. And 4T1 tumours treated with AIE/Biotin-M plus US irradiation induced the largest amount of HMGB1 release, which was demonstrated by the reduction of HMGB1 signal. These phenomena demonstrated that the combination of AIE/Biotin-M and US irradiation could potentiate the induction of tumor ICD, which would prominently activate immune response in vivo.
The DMAPs released from dying cancer cells could stimulate immature DCs to engulf tumor antigens and debris. Subsequently, the immature DCs migrate to the nearby lymph nodes, where they underwent maturation and exposed the processed pro-inflammatory cytokines to naive T cells, thus inducing a systemically innate immune response. Therefore, the antitumor innate immunity induced by the combination of AIE/Biotin-M and SDT was evaluated by analysing the maturation of DCs and secretion of pro-inflammatory cytokines. 4T1 tumor-bearing mice with tumor size of 80 mm3 were i.v. injection with AIE-M and AIE/Biotin-M at a concentration of 100 mg·kg− 1, respectively, once every 3 days three times. Due to the high tumor accumulation of AIE-M and AIE/Biotin-M at 2 h post-injection, at this time point, tumours were treated with US for 5 min or without US. After the last US irradiation for 48 h, single-cell suspension of tumor-draining lymph nodes was stained with anti-CD86, anti-CD80 and anti-CD11c for FCM analysis. As depicted in Fig. 5A, AIE-M and AIE/Biotin-M without US irradiation just slightly promoted DCs maturation (CD80+CD86+). However, in the presence of US, AIE-M and AIE/Biotin-M exhibited a high percentage of DCs maturation. Additionally, AIE/Biotin-M plus US significantly promoted DCs maturation (15.0%), which was consistent with the release of HMGB1 and CART.
The matured DCs could secrete pro-inflammatory cytokines, such as IFN-γ, TNF-α, and IL-6. Although AIE-M and AIE/Biotin-M in the absence and presence of US irradiation cloud increase the TNF-α, IFN-γ, and IL-6 levels (Fig. 5B) in peripheral blood of mice, AIE/Biotin-M plus US induced highest production of pro-inflammatory cytokines. In addition, the highest maturation of DCs and production of pro-inflammatory cytokines suggested that AIE/Biotin-M plus US could induce an immune response and showed great potential in antitumor immunity. Thus, single-cell suspension of tumours was stained with anti-CD8, anti-CD4 and anti-CD3 for FCM analysis of infiltrated T cells (Fig. 5C). In the absence of US irradiation, the percentage of tumor-infiltrating activated T cells (CD4 + and CD8 + T cells) slightly elevated in AIE-M and AIE/Biotin-M, due to the metal ions chelation of AIE micelles. And the level of T cells was significantly increased in US treated AIE-M and AIE/Biotin-M. The highest tumor-
infiltrating T cells in AIE/Biotin-M plus US was consistent with the DC maturation and pro-inflammatory cytokines release. These results revealed that AIE/Biotin-M plus US could activate the adaptive immune response and may potentially enhance anti-tumor therapy.
The in vivo therapeutic effect
Encouraged by the activation of systemically anti-tumor immune response by AIE/Biotin-M plus US, we further explored whether this combination could prevent breast cancer growth and metastasis in vivo. The mouse 4T1 tumor with poorly immunogenic, early metastasis and highly tumorigenic is analogous to human TNBC. Therefore, 4T1 tumor-bearing mice are regarded as an appropriate experiment model for simultaneously evaluating the therapeutic effect on inhibiting primary breast cancer growth and metastasis. 4T1 tumour-bearing mice with tumour volume 80 mm3 were randomly divided into six groups including PBS, PBS + US, AIE-M, AIE/Biotin-M, AIE-M + US and AIE/Biotin-
M + US. Mice were intravenously injected with AIE-M and AIE/Biotin-M at AIE micelles dose of 100 mg/kg, respectively. After i.v. injection for 2 h, tumors were treated with US (1 MHz, 2 Wcm− 2, 50% duty cycle) for 5 min or without US. As shown in Fig. 6A, merely US irradiation had no tumor inhibition compared to PBS treated mice, which validated that US irradiation has no damage to tissues. In the absence of US irradiation, AIE-M and AIE/Biotin-M showed slight tumor inhibition with average relative tumor volume (RTV) of = 8.5, 8.2 (Fig. 6A) and tumor inhibition rate (TIR) of = 33.1%, 36.3% (Fig. 6B) compared to PBS group due to the metal ions depletion in the tumor. Notably, in the presence of US irradiation, the generation of ROS and activation of immune response enhanced tumor inhibition of AIE-M with RTV = 4.3 and TIR = 64.4%. In the presence of US irradiation, Furthermore, the therapeutic ability of AIE/Biotin-M plus US irradiation on tumor tissues was conducted by histological, immunohistochemical, and immunofluorescent analysis of the tumor section at the end of therapy. To evaluate the apoptosis and/or necrosis, the excised tumors fixed, sectioned and stained to analyze H&E staining, TUNEL, and PCNA, Fig. 6F. The H&E staining results revealed the intact structure tumor cells in PBS and US treated tumors. Comparatively, tiny part of separated and sparse tumor cells was observed in AIE-M and AIE/Biotin-M group, which was lower than the AIE-M + US group. Notably, the most obvious damage of cells was observed in tumor tissue after being treated with AIE/Biotin-M plus US irradiation. Additionally, PCNA and TUNEL staining results further validated that AIE/Biotin-M plus US irradiation resulted in the weakest cell the TIR of AIE/Biotin-M was 1.3-fold higher than AIE-M due to tumor-targeting accumulation of AIE/Biotin-M triggered a strong generation of ROS and anti-tumor immunity. Meanwhile, weight measurements (Fig. 6C) and the photos (Fig. 6D) of tumors further validated that AIE/Biotin-M plus US effectively inhibited tumor growth. The six groups of mice were also measured for their body weights every two days, and no weight fluctuations were observed, demonstrating the biocompatibility of all treatments Fig. 6E. proliferation (weak red fluorescence in PCNA assay) and strongest cell apoptosis (strong green fluorescence in TUNEL assay). In addition to inhibition of tumor growth, reduction of tumor metastasis was also extraordinary significant in cancer therapy. As depicted in Fig. 6G, more significant amounts of tumor nodules were found in lungs and livers in PBS, US, AIE-M and AIE/Biotin-M than in AIE-M plus US irradiation, which was consistent with the histological examination. Compared to AIE-M plus US irradiation, AIE/Biotin-M plus US irradiation pronouncedly prevents liver and lung metastasis. Meanwhile, AIE/Biotin-M plus US irradiation could also result in the lowest vascular density and vascular endothelial growth factor (VEGF) in the tumor tissue (Additional file 1: Figure S5A). On the one hand, the generation of ROS during SDT could damage tumor vessels and VEGF. And the damage degree of vessel and VEGF was positively correlated with ROS production. On the other hand, AIE micelles with the ability of metal ions chelation could also inhibit angiogenesis and VEGF expression, which was demonstrated by the lowest concentration of Fe3+, Cu2+ and Zn2+ in AIE/Biotin-M treated tumour tissues, Fig. 7B. Although AIE/Biotin-M could induce metal ion depletion in tumors, negligible metal ions depletion was detected in other organs including liver, heart, lung, spleen, and kidney, Fig. 7B. Therefore, no apparent damage of cells was observed in liver, heart, lung, kidney, and spleen after tumor-free mice were i.v. injection of AIE-M and AIE/Biotin-M for 16 days demonstrated high biocompatibility of AIE/Biotin-M (Additional file 1: Figure S5B).