Design, fabrication, and characterization of HHBP nanosonosensitizers
The procedure for fabrication of HHBP nanosonosensitizer and the corresponding therapeutic mechanism are illustrated in Fig. 1. To endow the nanosonosensitizer with TME-responsive biodegradability, we introduced disulfide bond components into the framework of mesoporous silica to prepare organic-inorganic hybrid HMONs for the delivery of both 3BP and HMME. Typically, HMONs were prepared by employing an “ammonia-assisted selective etching” approach reported in the literature [37–39]. Initially, the monodispersed SiO2@MONs nanoparticles were synthesized through the co-hydrolysis of tetraethoxysilane and bis(3-triethoxysilylproyl) disulfide by employing cetyltrimethylammonium chloride as the structural-directing agents [40, 41]. As visualized by transmission electron microscopy (TEM) image, the as-synthesized SiO2@MONs exhibits a core/shell structure (Fig. S1). After etching in ammonia solution, the SiO2 core was removed and obtained the hollow HMONs of around 85 nm in size (Fig. 2a). Elemental mapping images show the presence of sulfur with the coexistence of silicon, carbon, and oxygen in HMONs (Fig. 2b-f), thus suggesting the formation of disulfide bond hybrid silsesquioxane framework within HMONs, as further proved by energy-dispersive spectrometry (EDS) analysis (Fig. S2). Meanwhile, the characteristic C signals in 13C cross-polarization solid-state NMR spectra and silicon resonances in the 29Si magic-angle spinning confirms the successful hybridization of disulfide bond into the framework of HMONs (Fig. S3). The amino-functionalized HMONs (HMONs-NH2) were prepared and then covalently modified with 3BP through the amide reaction between carboxyl groups of 3BP and amino groups of HMONs-NH2 (HMONs-3BP). The presence of peaks of amide group at 1623 (C=O stretching vibration) and 3278/cm (N-H stretching vibration) in Fourier-transform infrared (FT-IR) spectrum demonstrate the success formation of HMONs-3BP (Fig. S4). According to the N2 absorption-desorption isotherms analysis, the BET surface area of HMONs decreased from 481m2/g to 301 m2/g after the incorporation of 3BP (Fig. S5a), and the pore size decreased to about 3.8 nm (Fig. S5b). Even so, this HMONs-3BP was still highly suitable for encapsulation of hydrophobic molecules. Thus, organic sonosensitizers HMME was next loaded into the hollow cavity of the HMONs-3BP (designated as HMME@HMONs-3BP). Meanwhile, this nanosonosensitizer was further PEGylated through noncovalent interactions for improving the stability (yielding HHBP). It can be found from TEM images that the surface engineering and drug loading exhibited negligible change of morphology (Fig. 2g), while the hydrodynamic diameter of HHBP was increased to 142 nm (Fig. 2h), slightly larger than that of HMONs-3BP. After loading of HMME and PEGylation, the BET surface area was decreased to 206 m2/g and the mesopore size was also dramatically reduced to about 2.4 nm (Fig. S5). Also, the serial change of zeta potential further reconfirms the desirable synthesis in each step (Fig. S6). Furthermore, UV-vis spectra of HHBP show the characteristic absorption peaks at 398 nm (Fig. 2i), which originates from the HMME molecules. The loading capacity of HMME was found to be around 38% (HMME: HMONs-3BP, w/w) when the HMONs-3BP/ HMME feeding ratios is 0.5 (Fig. S7). These results suggested that HMONs could be efficiently loaded with HMME and modified with 3BP and be coated with PEG on the surface.
As the disulfide-bridged silsesquioxane framework of HMONs can be cleaved in the reductive TME [42, 43], the biodegradation behavior of HHBP was evaluated in simulated body fluids (SBF) mimicking intracellular redox conditions (GSH, 5 mM or 10 mM) in TME. From the TEM images, HHBP in SBF without GSH shows a certain extent but unconspicuous biodegradation during one week immersion in SBF. In contrast, HHBP are inclined to be gradually degraded in SBF solution containing 5 mM GSH and shows a time-dependent biodegradable behavior (Fig. S8). Especially, the biodegradation rate was significantly quicken and the nanoparticles were found to be entirely biodegraded in SBF solution containing GSH (10 mM) for 7 d. These results indicated the GSH-responsive biodegradability of this HHBP nanosized sonosensitizers.
In vitro HMME release and SDT effect of HHBP
Based on the unique GSH-sensitive biodegradation behavior of HHBP, we further evaluated their releasing performance and behavior under different GSH concentrations and pH values (Fig. 2j). As expected, the HMME release behavior from HHBP is highly dependent on its pH values as well as GSH concentrations. The percentage of HMME released after 24 h was less than 10% at pH 7.4 in absence of GSH, indicating the high stability of HHBP under physiological conditions. In contrast, the amount of released HMME dramatically increased to 26.2% at pH 5.5. Notably, the drug-releasing percentage sharply increased to ≈33.7% and ≈60.5% under GSH concentrations of 10 mM at pH 7.4 and 5.0, respectively. This finding can be attributed to the gradual biodegradation of the HMONs framework induced by GSHinduced cleavage of the disulfide bond. Excitingly, the release rates are further enhanced and approximately 76.5% HMME release was observed after 24 h upon exposure to US irradiation, which may be due to the dissociation of HMME from HMONs caused by the mechanical/cavitation effects of US [2, 44, 45]. Taking into account the tumor environment is distinguished by mild acidic and high GSH concentration, the pH/GSH/US tri-stimuli-responsive HHBP are expected to deliver hydrophobic sonosensitizers for substantially enhanced SDT efficacy.
Subsequently, the SDT performance of HHBP was evaluated based on the presence of HMME, by using 1,3-diphenylisobenzofuran (DPBF) as a 1O2 probe to measure the ROS production under US irradiation (Fig. 2k). After irradiation with the US, the production of 1O2 was further improved, as evidenced by the attenuated UV absorption peak of DPBF at 398 nm when the US irradiation time increased. Additionally, electron spin resonance (ESR) with the spin traps of 2,2,6,6-tetramethylpiperidine (TEMP) were also acquired. According to ESR spectra (Fig. 2l), the strong 1O2 signal (1:1:1) was detected in the HHBP + US group, while no obvious ESR signal could be observed in the HHBP alone group and PBS group, even upon irradiation with US (1.0 W/cm2, 1.0 MHz, 50% duty circle, 1 min). The above results indicate that HHBP could be an ideal nanosonosensitizer for US-triggered ROS generation.
Intracellular uptake and hypoxia alleviation of HHBP
Inspired by the afore-mentioned results, we next evaluated the cellular uptake and O2-consumption reduction ability of HHBP in 4T1 cells. Initially, the in vitro cytotoxicity of HMONs-PEG was determined by a cell counting kit-8 (CCK-8) assay. HMONs-PEG shows no significant cytotoxicity to various cancer cells in vitro (Fig. 3a), including HUVEC, 4T1, A375, and A549 cells, even at high concentrations of 400 µg/mL, demonstrating the relatively good biocompatibility of HMONs-PEG. In order to evaluate the intracellular endocytosis behavior, the cellular uptake efficiency of HHBP in cells was investigated. Confocal laser scanning microscopy (CLSM) images notices a time-dependent endocytosis process, as evidenced by increased FITC signals at extended co-incubation durations (1, 2, 4, and 8 h) of HHBP with 4T1 cancer cells (Fig. 3b). More excitingly, after further irradiated with US, a stronger green fluorescence was found in 4T1 cells in the presence of HHBP. In addition, flow cytometry analysis was also performed to measure the fluorescence intensity of FITC (Fig. S9). The cells treated with HHBP exhibit high uptake efficiency as the incubation time prolonged and the US stimulation could enhance the cellular uptake of nanoparticles, which are consistent with the CLSM observation.
As an excellent respiration inhibitor, 3BP is expected to be a hypoxia ameliorator for reducing O2 consumption, which is the essential condition for amplifying SDT.[46] To verify it, the expression of and hexokinase (HK)-II, which involves in the first stage of cellular respiration, were evaluated in the cells after treated with different formulations by western blotting analysis (Fig. 3c). Notably, negligible change of HK-II and HIF-1α levels was detected in 4T1 cell after incubated with HMME@HMONs-PEG nanoparticles, while HHBP with or without US irradiation could markedly reduce the protein expression levels (Fig. S10). Arguably, the introduction of 3BP played a vital role in the down-regulation of HK-II and HIF-1α. These results proved that the inhibition of HK-II used by such HHBP exposure could inhibit cellular respiration, which would cause changes on cellular oxygen consumption. Furthermore, the effect of HHBP under US irradiation to the mitochondrial dysfunction was evaluated by measuring the variation of mitochondrial membrane potential, with commercial JC-1 dye. We found that the 4T1 cells treated with HHBP with or without US irradiation displays strong green fluorescence (Fig. 3d), in marked contrast to the prominent red fluorescence after treated with PBS or HHP, indicating respiration is further suppressed.
In vitro augmented SDT efficacy of engineered HHBP nanosonosensitizer at cellular level
Inspired by the excellent O2-economization properties of HHBP nanosonosensitizer, the intracellular ROS generation and in vitro augmented SDT efficacy were further assessed. The intracellular ROS levels was visualized by a of 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) probe, which could be oxidized into green fluorescent, 2’,7’-dichlorofuorescin (DCF) in the presence of ROS [47, 48]. As revealed in Fig. 3e, negligible fluorescence was detected in PBS, HMME@HMONs-PEG, and HHBP group, while the cells treated with the latter two under the US irradiation display conspicuous green fluorescence, indicating a strong ROS generation. Further quantitative flow cytometric results summarized in Fig. 3f demonstrated that the cellular fluorescence intensity of HHBP + US treatment was significantly higher than that of other treatments. As a result, HHBP is able to induce plentiful ROS production under US irradiation, shows great potential for SDT against tumors. Encouraged by the prominent ROS generation performance of HHBP, in vitro augmented SDT effect was assessed on 4T1 cell by CCK-8 assay. As expected, the viabilities of the cells after treatment with “HMME@HMONs-PEG + US” and “HHBP” decreased to 62.5% and 44.9% at a HMME concentration of 38 µg/mL, respectively (Fig. 4a). In contrast, much lower cell viabilities were detected after treated with HHBP at corresponding concentrations under US irradiation, suggesting the excellent synergistic effect of SDT and 3BP. Similar results were also revealed by calcein AM/PI stain assay (Fig. 4b). Moreover, the treatment of HHBP under US irradiation could also achieved effective inhibition effect on diverse tumor cells including A375 and A549 cells with a strong concentration-dependent cytotoxicity (Fig. S11). Significantly, almost no green fluorescence could be observed when the cells were treated with HHBP and US irradiation. In addition, the apoptosis effect induced by the synergistic effect was evaluated by Annexin V-FITC/PI based flow cytometer assay (Fig. 4c). The apoptosis/necrosis rate of cells treated with “HHBP + US” was 69.7%, which was much higher compared with other treatments. Taken together, the HHBP nanosonosensitizer could reduce O2 consumption for enhanced SDT, promising in improving the therapeutic outcomes cancer treatment.
Pro-death autophagy induced by HHBP promoted cancer cell apoptosis
Here, it should be noted from the vitro therapeutic results that HHBP treatment without US irradiation can also acquire moderate inhibitory effects. Therefore, 3BP implicated an essential role in inhibiting cell viabilities. In previous studies, 3BP can suppress both mitochondrial respiration and glycolysis to lead to starvation-induced autophagy [46]. To further verify the hypothesis that 3BP can induced pro-death autophagy, we evaluated the expression of autophagy-related protein (LC3 and p62) in 4T1 cells receiving different treatments by Western blot assay. Notably, highest cellular autophagy levels were detected when treated with HHBP plus US irradiation (Fig. 4d), which was proved by the p62 inhibition as well as the LC3-II/LC3-I ratio elevation. Quantification results shown on Fig. S12 further indicated the LC3-II/LC3-I ratio increased by 98-fold after the HHBP + US treatment comparing with control cells, while the expression of p62 reduced to 1.5%. These phenomena demonstrate that the synergistic effect of 3BP and SDT could effectively induce autophagy in 4T1 cells. Furthermore, immunofluorescence staining was performed to characterize the autophagy level by visualized monitoring of the LC3 punctate dots (Fig. 4e). Both HHBP alone and HMME@HMONs-PEG + US treatments exhibit green significant fluorescence signals, which are attributed to the autophagy induced by 3BP and ROS, respectively. When combined with HHBP and extra US irradiation, the green fluorescence was significantly enhanced. The formation of acidic vesicular autophagosomes during autophagy process was examined by utilizing the monodansylcadaverine (MDC) staining (Fig. 4e). It can be observed that the treatment of HHBP incubation plus US exposure resulted in most strong green fluorescence of acidic autophagosomes compared to the cells treated with HHBP and HMME@HMONs-PEG + US. To observe the autophagosomes more intuitively, the 4T1 cells receiving different treatments were observed by TEM (Fig. 4f). Unequal in quantity of autophagosomes could be detected in all treatments except the control group, which indicated that 3BP or SDT-induced ROS could trigger cell autophagy. Meanwhile, HBBP incubated 4T1 cells exposed by US represented more autophagic vesicles than that in other treatments. These results suggested that the synergistic effect can active the excessively autophagy, which could also promote cell death. Concomitantly, the ATP level in 4T1 cells was investigated. As shown in Fig. S13, HMME@HMONs-PEG treatment had no obvious influence intracellular ATP level as compared to the control group. However, both 3BP and HHBP dramatically reduced the ATP to 17.8% and 20.2%, respectively, suggesting the depletion of ATP induced by 3BP could significantly elevated the autophagy level. These results agreed well with the therapeutic evaluations described above and offered reliable evidence that the improved anticancer activity of HHBP-mediated augmented SDT could be ascribed to the synergistic effect of apoptosis and autophagy.
In vivo biodistribution of the HHBP nanosonosensitizer
The in vitro outstanding properties of the nanosonosensitizer encouraged us to further assess the therapeutic performance on 4T1 breast tumor xenografts in nude mice. For tracking the in vivo distribution and tumor accumulation behavior of HHBP, a near infrared dye, indocyanine green (ICG) was loaded to HHBP through a simple mixing method. From the in vivo fluorescence images (Fig. 5a), the fluorescence signals in the tumor site were increased upon prolonging time, with the maximum signal at 12 h post-injection, which is also maintained at 16 h, and then declined due to the degradation of ICG. Correspondingly, the tumor and main organs of mice were excised at 24 h for ex vivo imaging. Obviously, with ICG-HHBP-treated mice showed strong ICG fluorescence in the tumor (Fig. S14). Also, strong fluorescence signal was detected in liver, which may be attributed to the specific uptake of mononuclear phagocyte system [49]. These results suggested the favourable tumor accumulation performance of HHBP.
In vivo alleviation of hypoxia
On the basis of the above in vitro findings, HHBP is expected to be a hypoxia modulator for reducing O2 consumption, which is beneficial to overcome tumor hypoxia and improving SDT efficiency. Before testing the antitumor efficacy, the ability of HHBP to ameliorate tumor hypoxia was evaluated by hypoxia inducible factor-1α (HIF-1α) immunofluorescence staining assay. As observed under the fluorescence microscopy (Fig. 5b), extensive green fluorescence was observed in PBS and HMME@HMONs-PEG group, confirming the overexpression of HIF-1α under hypoxic conditions. In contrast, the indicators of HIF-1α were obviously downregulated after treatment with HHBP with or without US irradiation, which proved that 3BP could effectively relieve tumor hypoxia.
In vivo antitumor efficacy enabled by HHBP nanosonosensitizer
To investigate whether the HHBP-mediated augmented SDT strategy was applicative to in vivo experiments, the therapeutic performance of HHBP against 4T1 tumor-bearing mice model was performed. Mice were randomly divided to six groups (n = 5): (Ⅰ) PBS, (Ⅱ) HMME@HMONs-PEG, (Ⅲ) HMME@HMONs-PEG + US, (Ⅳ) HHBP, and (Ⅴ) HHBP + US. The injected dosage was set as HMME 8 mg/kg and 3BP 3.4 mg/kg (100 µl). The tumor site of mice in Group Ⅲ and Ⅴ were exposed to US (1.5 W/cm2, 1.0 MHz, 50% duty circle) for 5 min and twice as much US exposure on days 1 and 4, respectively. To examine the ROS level at tumor sites, the tumors after different treatments were collected after US irradiation for dihydroethidium staining (Fig. 5c). It was uncovered that both HMME@HMONs-PEG and HHBP under US exposure could improve ROS levels, both of which were obviously higher than other treatments. More specifically, higher ROS level could be achieved by HHBP + US treatment, which validated that 3BP could relieve tumor hypoxia for cause tremendous ROS generation via the amplification of HMME-initiated sonodynamic effect. Following the treatments, tumor volumes (Fig. 6a) and body weights (Fig. 6b) of the mice monitored within two weeks. Rapid tumor growth displayed in PBS and HMME@HMONs-PEG treated group, while HHBP and HMME@HMONs-PEG plus US exposure led to moderate growth of tumors (inhibition rate: 40% and 28.5%, respectively). More excitingly, the tumors on the mice after treated with HHBP and US exposure were the most effectively suppressed (inhibition rate: 89.1%). The corresponding images of representative tumors on day 14 in different treatments further confirmed that the HHBP + US group realized an intelligent antitumor effect (Fig. 6c). In addition, there were negligible body-weight loss of mice in any of the groups during the treatment (Fig. 6b), demonstrating the low adverse effects of all formulations in vivo. The above desirable therapeutic efficacy might be benefited from the excellent synergistic therapeutic effect, including the 3BP-mediated hypoxia modulation, HMME-based augmented SDT, as well as 3BP-induced excessive activation of autophagy.
To further reveal the performance mechanism of HHBP, the apoptosis and autophagy level of the collected tumors from all groups were evaluated. Compared to other treatments, most significant large-area histological damage regions from H&E staining, most significant inhibition of cell proliferation from Ki-67 assay, as well as the highest evident apoptosis of tumor cells from TUNEL staining were observed in the “HHBP + US” treatment group (Fig. 6d). Such results are consistent with the foregoing results of in vivo anticancer experiment. Subsequently, we also checked the autophagy levels in tumor sections after different treatments by immunofluorescence imaging staining of LC3 (Fig. 6e). It was found that both HMME@HMONs-PEG + US and HHBP alone could improve autophagy levels, according to the green fluorescence of LC3 puncta showed in immunofluorescence images. Consistent with the in vitro data, the HHBP group showed the strongest green fluorescence after US irradiation, which represented an excessive activation of autophagy by 3BP and SDT.
Last, but not the least, the safety assessment of HHBP on mice was performed. H&E sections of the collected major organs did not observe obvious inflammation after injection with HHBP for 3, 7, and 14 days (Fig. S15), indicating the favorable biocompatibility of HHBP. Concomitantly, the blood sample was collected from healthy female Balb/c mice at the different times post-injection of HHBP for blood biochemistry and blood routine examination. No obvious changes detected in relation to blood routine indexes and liver/kidney function associated biomarker induced by HHBP compared to the control group (Fig. S16), partly indicating ignorable systemic toxicity. These results indicated that the fabricated HHBP may augment SDT efficacy with causing relatively lower systematic toxicity in vivo.