Characteristic of nanoparticles
ZTC was prepared using zeolitic imidazolate framework 8 (ZIF-8) coloaded with ZTC and Ce6. The final nanoparticle called [email protected] was prepared by extruding and wrapping cytomembrane fragments of AGS cell. TEM images showed that ZTC had the same structure as ZIF-8 (Fig. 1a and b) with a uniform size (Fig. 1d and e). The [email protected] nanoparticle was covered with cytomembrane as seen through the TEM (Fig. 1c). Additionally, the surface was observed to be slightly smoother than ZTC (Fig. 1b and d). The size was also bigger than ZTC (Fig. 1d and f), with an increase in size reported from 175 nm to 253 nm. Besides, the comparison with the surface potential before modification of AGS cytomembrane showed a reversal from positive 13.8 mV to negative –16.4 mV, indicating successful coating of AGS cytomembrane (Fig.1h). This result was consistent with the gel electrophoresis result (Fig. 1g). Also, the stability of [email protected] was evaluated, where the results showed that the size did not change in ultrapure water within a week (Fig. 1i), indicating that the nanoparticle was stabilized.
The absorption peaks of TPZ (470 nm) and Ce6 (400 and 660 nm) were presented in the absorption spectrum of [email protected] (Fig. 1j), indicating successful loading of the nanoparticle with TPZ and Ce6. Simultaneously, the loading capacities of Ce6 and TPZ were calculated, which were found to be 77.57±0.48% and 53.86±3.48%, respectively. The results illustrated that the frame ZIF-8 could co-load both TPZ and Ce6 for the cooperative therapy.
DPBF is a typical analytical reagent of ROS and is used to analyze the production of ROS. Compared to the control groups, the remaining DPBF in the [email protected] group irradiated with the US was found to be significantly lower (Fig. 1k), indicating that [email protected] upon US irradiation could produce much more ROS from Ce6.
The CCK-8 assay was conducted to explore the best working concentration of this nanoparticle. When the loading content of TPZ reached 8 mg, the viability of the cells was inhibited down to 40% (Fig. 2a), determining the quantity of TPZ to be loaded as 8 mg. Moreover, ZIF-8 and Ce6 showed no obvious cytotoxicity to AGS cells (Fig. 2b). The cytotoxicity of nanoparticles was found to be weaker than that of a single TPZ drug (Fig. 2b), suggesting a weakening of the toxicity of the single drug by the nanoparticle. Also, no cytotoxicity was observed with the concentration of nanoparticles being up to 30 µg/mL (Fig. 2b). Thus, the working concentration of the nanoparticle was chosen as 30 µg/mL.
The hemolysis assay was conducted to analyzed the biocompatibility of [email protected] The hemolysis rate below 10% was considered safe for intravenous injection.The hemolysis rate was observed to be less than 1% when the concentration of [email protected] used was up to 300 µg/mL, which was an acceptable range for intravenous administration (Fig. 2c and 2d), indicating good biocompatibility.
The drug-releasing ability of TPZ and Ce6 from [email protected] was evaluated in PBS at two different pH. Comparing with pH 7.4, the release rate was increased in the solution having pH 5.5 (Fig. 2e and f) due to its pH-responsive decomposition ability. This illustrated an increased amount of release of Ce6 and TPZ in the PBS solution with pH 5.5. Incubation for 6 h at pH 5.5 released approximately 81.49±1.58% of loaded TPZ and 62.20±0.55% of loaded Ce6 from the [email protected], and these values were much higher than the values obtained at pH 7.4. Furthermore, the releasing ability of Ce6 from [email protected] was evaluated with/without the US. It was found that the release rates were higher when irradiated with the US than the ones without the US irradiation (Fig. 2e). After the treatment with the US at pH 5.5, 78.18 ±0.86% of loaded Ce6 was released from [email protected] Thus, in cooperation with the US, the pH responsiveness of [email protected] was applied to release drugs in an acidic solution to attain a combined target treatment.
The results of the cell fluorescence showed that compared to the incubation at pH 7.4, AGS cells showed higher fluorescence intensities at pH 5.5 (Fig. 3a and c). After the addition of cytomembrane of AGS, stronger fluorescence intensities were presented in the [email protected] group, comparing wirh the ZTC group. Further, the signal intensities from AGS cells wrapped with cytomembrane of other species cells (4T1, mouse breast cancer cell) showed weaker signals than the cells wrapped with a homologous tumor cytomembrane (Fig. 3a and c). The results of the flow cytometer were consistent with the results of the cell fluorescence (Fig. 3b and d). Nanoparticles can be better degraded and released in the site of the microenvironment, which is similar to the tumor acidic microenvironment, Also, free Ce6 can easily enter the cells in such case. Additionally, because of the homologous cytomembrane, more nanoparticles can be taken up by the cells. All of these indicate that [email protected] especially recognizes homologous tumor cytomembrane and makes use of the tumor acidic microenvironment to target the release of the drug and achieve anti-tumor function.
In vitro ROS/Hypoxia assay
Intracellular ROS levels were evaluated by DCFH-DA, whose green fluorescence was enhanced with the increase in ROS level inside the cells. As presented in Fig. 4a and e, the intensity of ROS green fluorescence in the group irradiated by the US ([email protected]+US) was found to be much higher than that found in ZC, ZTC, and [email protected] groups without the US (P < 0.0001), which indicated that Ce6 irradiated by the US released ROS. The reason behind this could be attributed to Ce6 being a sonosensitizer, which may produce ROS after the treatment with the US.
Next, the ability to induce hypoxia by ROS was evaluated by the hypoxia reagent. The Image-iT™ Green Hypoxia Reagent is an end-point assay reagent whose signal increases with the reduced oxygen levels. The green fluorescence is enhanced with the increase in hypoxia level. Our results showed that the green fluorescence in the CON group was weaker than that of the ZTC (p = 0.0068), [email protected] (p = 0.0034), and [email protected]+US (P<0.0001) groups (Fig. 4b and f). Meanwhile, the strongest green fluorescence was seen in the [email protected]+US group, which was even stronger than that of the [email protected] group (P<0.0001) (Fig. 4b and f). This can be explained by the increased hypoxia in cells released by Ce6 due to the accumulation of ROS. Also, the release of ROS and increased hypoxia in cells is enhanced by the US.
HIF-1α is considered as a key mediator of signal in poorly oxygenated areas and can stay steady under hypoxia environment [32, 33]. In normoxia conditions, HIF-1α is always inactivated and degraded hence, it is hardly detected . HIF-1α can stay stabilized in poor oxygen conditions and can be translocated to the nucleus responding rapidly to oxygen deprivation . Further, IF and WB of HIF-1α were performed to demonstrate the reduction of intracellular hypoxia by ROS consumption. With an increase in hypoxia level, the green fluorescence of HIF-1α could be strengthened. It was found that the green fluorescence observed in the [email protected]+US group was stronger than any other group, i.e., CON, ZC, ZT ZTC, and [email protected] groups (P<0.0001) (Fig. 4c and g), showing that the hypoxia level of the [email protected]+US group was the strongest. In Fig. 4d and h, the levels of HIF-1α protein in the [email protected]+US group were found to be much higher than that of the CON group (P<0.0001) and [email protected]+US group (P<0.05), which was in accord with the results of IF. These results proved that the ROS released from Ce6 could induce intracellular hypoxia, which provided a hypoxic environment to TPZ.
In vitro therapeutic effect
To evaluate the killing effect of nanoparticles, Calcein/PI staining and apoptosis assays were conducted. Fig. 5a showed that the red fluorescent intensities from dead cells in the ZTC group were stronger than that found in the ZT and ZC groups (P<0.05), indicating that the efficiency of nanoparticles was better in combination with Ce6 and TPZ than that of a single drug. Meanwhile, the red fluorescence intensities of the cytomembrane target group ([email protected]) were higher than those groups without cytomembrane (ZTC) (P<0.05). This was because the homologous AGS cytomembrane specifically increased the uptake of nanoparticles by AGS cells and enhanced the killing effect on tumor cells. Also, after the US treatment, a much stronger red fluorescence was seen in most of the cells. The quantitative analysis of fluorescence presented that the therapeutic effect of the SDT treatment group ([email protected]+US) was found to be higher than that of the group without the SDT ([email protected]) (Fig. 5a and c) (P<0.05).
Similar results were shown by the apoptosis assay (Fig. 5b and d). The percentages of apoptotic cells in CON, ZC, ZT, ZTC, [email protected], and [email protected]+US groups were approximately 8.13%, 17.40%, 15.49%, 24.10%, 49.87%, and 72.77, respectively. There was a significant difference observed between ZTC and ZT/ZC. A higher rate of apoptosis was observed in [email protected] compared to that of the ZTC. The highest apoptosis rate was demonstrated in the group treated with US ([email protected]+US). All these results illustrated that targeting the drug along with SDT treatment could enhance the killing effect on tumor cells.
In vivo biocompatibility research
To verify the safety in vivo, we evaluated the biocompatibility of nanoparticles. Fig. 6a shows that there were no significant differences in the counts of white blood cell (WBC), red blood cell (RBC), hemoglobin (Hb), and platelet (PLT) between the groups observed on 7/30 d and in the control group, illustrating that there was no significant blood toxicity found in [email protected] in vivo. Meanwhile, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and blood urea nitrogen (BUN) were detected to study the presence of any liver and kidney damage. As shown in Fig. 6a, no significant differences were observed in these indexes in the three groups. Also, to confirm whether nanoparticles can induce organ damage, the H&E staining of organs was compared among the three groups. The results demonstrated that no apparent damage or pathological changes were observed in the experimental groups, which showed consistency with the control group (Fig. 6b). Hence, these results presented that [email protected] had good biocompatibility with no apparent toxicity in vivo, indicating it to be a potential nanoparticle for in vivo anti-tumor experiments.
In vivo SDT therapy
Nude mice bearing AGS tumors were used as the model to investigate the anti-tumor effect of SDT therapy of [email protected] As shown in Fig. 7b, there was no significant loss of body weight in mice among all six groups during the treatment, which demonstrated that the nanoparticles had good biocompatibility with no evident toxicity to mice. It was observed that the growth of tumors treated with PBS was a little more rapid than those treated with ZC and ZT (Fig. 7c), indicating that single-drug therapy had limitations. Simultaneously, Fig. 7c shows that the growth of tumors was slightly slower in the group treated with nanoparticles combined with Ce6 and TPZ than that of a single drug, which showed that combined therapy worked relatively better than a single drug that had limited efficiency. However, the growth of the tumor in the targeting group ([email protected]) was further reduced, and the inhibition of tumor growth was found to be stronger than the previous three groups, but it still could not reduce the growth of the tumor completely. The tumor growth was inhibited in the [email protected] mice treated with the US irradiation compared to those without the US, indicating a significant anti-tumor efficiency by the US. Regarding the tumor inhibition ratio (Fig. 7d), the inhibition rate reached up to approximately 87% in the [email protected]+US group, which was higher than any other group, indicating the same results as the tumor volume. The reasons behind these results might be explained as follows: firstly, the nanoparticles encapsulated with homologous tumor cytomembrane enhanced its targeting ability and were recognized easily by tumor cells; hence, more nanoparticles could enter tumor cells and perform the anti-tumor function. However, without US irradiation, Ce6 could not be activated completely, and the function was limited. Secondly, after irradiation with the US, the accumulation of ROS released by Ce6 could aggravate hypoxia in the tumor microenvironment and kill tumor cells. Finally, hypoxia further activated the anti-tumor effect of TPZ and induced the death of tumor cells. These results demonstrated that [email protected]+US could efficiently inhibit tumors and exert anti-tumor function.
Further, H&E staining of the tumor was performed to evaluate the therapeutic efficacy. An obvious necrotic tissue was observed in the tumors of mice treated with the [email protected]+US group compared to the other groups (Fig. 7e). Further, the TUNEL staining was performed to observe the tumor injure in mice among the different groups (Fig. 7e). The results indicated that there was a small number of apoptotic cells observed in the tumors of PBS, ZC, ZT, and ZTC groups. In the [email protected] group, many apoptotic cells were seen, but the number was less than that of the [email protected]+US group. These results were in accord with the results of H&E staining.
[email protected] induced ROS contributed to AGS pyroptosis
Some studies reported that the ROS contributed to apoptosis and pyroptosis[36, 37].Pyroptosis is different from cell apoptosis and is an inflammatory form that mediates the programmed death of cells.It is activated by caspase-1/4/5/11 and can lead to cell damage, including fragmentation of chromatin, cell swelling, lysis of plasma membranes, and release of intracellular proinflammatory contents[39, 40].It is well known that caspase-1, NLRP3 (NACHT, LRR, and PYD domain-containing protein 3), and ACS molecules can induce pyroptosis in tumor cells. Caspase-1 is the activator of pyroptosis, which can induce the release of inflammatory factors, i.e., interleukin-1 beta (IL-1β) and interleukin-18 (IL-18).ACS comprises a caspase recruitment domain and a pyrin domain and is the adaption protein of NLRP3.The NLRP3 inflammasome, an inflammatory protein complex, consists of the sensory molecule NLRP3, ACS, and caspase-1, which is the most characterized inflammasome[43, 44].It is also well known that ROS is an activator of NLRP3 inflammasome[45, 46].
To determine if the nanoparticles could induce pyroptosis, the levels of these proteins were detected by western blotting. The [email protected] groups exhibited higher levels of caspase-1, NLRP3, and ACS compared to that of the control groups (Fig. 8a and b, all P-value<0.05). Comparing with the control and [email protected] groups, the levels of caspase-1, NLRP3, and ACS in the [email protected]+US group were found to be significantly higher (Fig. 8a and b, all p-value<0.05). It demonstrated that caspase 1, caspase 3, GSDMD, NLRP3, and ACS were all upregulated after being irradiated by the US. These results showed that the ROS produced by the ZTC[email protected] group treated with the US could induce pyroptosis in AGS cells, which enabled it to play the anti-tumor function.
Cells undergoing pyroptosis show distinct morphological features. Deron R. Herr et al. proved that the most prominent pyroptotic cells represented cell swelling, the retraction of cellular processes, and the emerging of pores in the cell surface . AGS cells were also evaluated for detailed surface morphology by phase-contrast microscopy (Fig. 8c and d) after the [email protected]+US treatment. Untreated AGS cells appeared normal under microscopy showing characteristics such as extended processes among the cells (Fig. 8c). After the treatment with [email protected]+US, it was found that the loss of processes and cell swelling manifested as a significant increase in the cell size (Fig. 8d). Further, the surface morphology was observed by the SEM, and the most prominent treated cells were found in the numerous pits or pores of different sizes across the cell surface (Fig. 8f), which was correlated with collapsing of the structure and flattening of the cell shape (Fig. 8e). Simultaneously, it also presented a rounded morphology due to the complete retraction of cellular processes (Fig. 8e). Hence, proving that our nanoparticles could induce cell pyroptosis.