In situ synthesized nanozyme for photoacoustic-imaging-guided photothermal therapy and tumor hypoxia

In situ
synthesized nanozyme for photoacoustic-imaging-guided photothermal therapy and tumor hypoxia relief

Original author：Chaoyi Chen,1
Yuwen Chen,1
Xuanhao Wang,1
Lulu Zhang,
Yan Luo,
Qingshuang Tang,Yuan Wang,
Xiaolong Liang,∗
and
Cheng Ma*

“
In this study, we present an innovative in situ synthesized nano-enzyme designed and synthesized based on intelligent nanosystems (ISSzyme) of Prussian blue (PB) precursor.ISSzyme is capable of interacting with glutathione to synthesize PB nano-enzymes at tumor-specific locations. This novel nano-enzyme not only enables tumor-specific photoacoustic imaging (PAI) and photothermal therapy (PTT), but also effectively reduces the false-positive signals of PAI and potential side effects of PTT. Meanwhile, ISSzyme exhibits catalase activity, which can alleviate tumor hypoxia and thus inhibit tumor metastasis. In addition, the in situ synthesized PB nanoenzymes do not cause liver damage due to their low accumulation in the liver.The study of ISSzyme provides new ideas for designing the next generation of artificial enzymes and lays the foundation for opening up a variety of new biomedical applications."

01

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Synthesis background and principle

Nanoenzymes have attracted much research attention in the past decade for their enzyme-like activities, and they have demonstrated superiority over natural enzymes in terms of cost, catalytic activity, diverse enzyme-like activities, and stability. As a new generation of artificial enzymes, nanoenzymes have great potential for biosensors and biomedical applications. However, non-specific accumulation of most nanoparticles in metabolic organs leads to inefficient drug delivery and may damage normal organs. For example, accumulation of nanoenzymes in the liver, which is rich in phagocytes and 50-200 nm pores, may lead to hepatotoxicity, disruption of redox homeostasis, and negatively affect tumor therapy. In addition, trapping of nanoenzymes in the circulation may lead to false signals during diagnostics and damage normal tissues. Therefore, it is particularly important to design controlled nanoenzymes with high efficiency and biosafety.

Prussian Blue nanoenzymes have potent antioxidant activities, including peroxidase-like, catalase-like and superoxide dismutase-like activities, which are essential for regulating oxidative stress in disease. They can decompose hydrogen peroxide to generate oxygen at the tumor site, alleviating tumor hypoxia and inhibiting metastasis. Meanwhile, Prussian Blue nanoenzymes remove excess reactive oxygen species (ROS) from the body and are used in the treatment of ROS-related diseases. Due to its excellent near-infrared (NIR) absorption and photothermal conversion efficiency, Prussian blue nanoparticles are also widely used as photothermal agents for photothermal therapy (PTT). Importantly, Prussian blue nanoenzymes can reduce inflammation during PTT and prevent damage caused by excessive hyperthermia. However, Prussian blue nanoenzymes do not function to specifically bind to tumors, and conventional PA contrast agents may produce false-positive signals during the diagnostic process. Therefore, designing PA contrast agents that can specifically bind to tumors and switch the response is a commonly used strategy. In the tumor microenvironment (TME), glutathione (GSH) is the most abundant endogenous active small molecule in cells and tissues, which plays an important role in life activities and maintenance of redox balance. Based on this, researchers have designed ISSzyme, a nanomedicine for cancer therapy and tumor hypoxia mitigation.PB nanoenzymes can be synthesized on-site in vivo when ISSzyme reacts with GSH, which is highly expressed in tumors. Intratumorally generated PB nano-enzymes have peroxidase-like activity and can decompose H2O2 to generate O2 to alleviate tumor hypoxia and inhibit metastasis.

Figure 1 ISSzyme-specific combination of tumor-guided photothermal therapy (PTT)

and the process of relieving tumor hypoxia is shown schematically.

02

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In vitro responsiveness verification

In order to investigate the responsiveness of ISSzyme to GSH, the absorbance and PA amplitude of ISSzyme solutions were measured in the presence of different concentrations of GSH. The results showed that the absorbance and PA amplitude of ISSzyme at 700 nm increased linearly with increasing GSH concentration, while the PA amplitude at 990 nm remained essentially constant. This suggests that it is possible to ratiometrically image the PA amplitude ratio at 700 nm and 990 nm (PA700/PA990), which increases significantly with increasing GSH concentration, showing a 4.87-fold increase from 10 ± 3 at zero concentration to 48.7 ± 0.9 at saturation value. In addition, in the in vitro photothermal capacity assessment experiments, the temperature increase of ISSzyme solution in the presence of GSH by NIR laser irradiation showed a significant correlation with the GSH concentration. This suggests that ISSzyme is able to respond efficiently to GSH and shows significant property changes in vitro through ratiometric PA imaging and photothermal effects.

Figure 2

Properties of ISSzyme enzyme

(A and B) Representative TEM images of ISSzyme (A) and GSH-treated ISSzyme (B). (C) EDS elemental mapping of K and Fe in ISSzyme before and after GSH co-culture. (D) Photographs of ISSzyme solution after addition of GSH (0-8 mM, 5 min). (E) UV-Vis absorption spectra of ISSzyme at different concentrations of GSH addition. (F) Variation of specific absorption signal (Abs700/Abs990) of ISSzyme with GSH concentration. (G) PA images of ISSzyme in the presence of different concentrations of GSH. Samples were recorded at 700 and 990 nm and are shown in green and red, respectively. (H) Quantification of the ISSzyme ratio PA signal (PA700/PA990) varying with GSH concentration. (I) In vitro IR thermograms of ISSzyme treated with GSH (from 0 to 20 mM) before and after irradiation (808 nm, 0.8 W cm-2, 5 min). (J) Corresponding photothermal heating statistics in (I). (K) Specific absorption signals (Abs700/Abs990) of ISSzyme in the absence (control) or in the presence of different amino acids (10 mM). (L) ICP-OES measurements of ISSzyme. (M) Oxygen production time course of ISSzyme under different treatment conditions.

03

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In vitro cytotoxicity and hypoxia alleviation experiments

Experiments were conducted to evaluate the photothermal treatment effect of ISSzyme on 4T1 breast cancer cells. The results showed that the viability of 4T1 cells gradually decreased with the increase of ISSzyme concentration. When the ISSzyme concentration reached 200 mg mL-1, the cell viability was significantly reduced to 8.54% after NIR laser irradiation. Further experiments showed that the cell viability after ISSzyme treatment was negatively correlated with the irradiation power density and time, indicating that ISSzyme has a rapid and stable photothermal conversion ability. In vitro assessment of anticancer ability showed that neither irradiation treatment alone nor ISSzyme treatment had a significant anticancer effect, but cell viability was significantly reduced when the two were combined. Live-dead cell staining and flow cytometry results further demonstrated the effective photothermal therapy (PTT) ability of ISSzyme and its effect on apoptosis. In addition, ISSzyme treatment alleviated tumor hypoxia and inhibited tumor metastasis, as evidenced by a decrease in the invasiveness of tumor cells cultured under hypoxic conditions, as well as a significant reduction in the relative number of metastatic cells. In summary, ISSzyme, as a photothermal therapeutic agent, is able to effectively inhibit tumor growth and metastasis in vitro, and combined with irradiation treatment exhibits enhanced anticancer potential by mechanisms involving photothermal conversion, on-site production of oxygen, and induction of apoptosis.

Figure 3.

Results of in vitro ISSzyme-mediated cytotoxicity and hypoxia alleviation experiments (A) Survival of 4T1 cells after near-infrared irradiation at different concentrations of ISSzyme (n = 4). (B) Survival of 4T1 cells under different irradiation conditions after treatment with the same concentration of ISSzyme (n = 4). (C and D) Survival rate (C) and calcein-AM/PI staining (D) of 4T1 cells with different treatments (ISSzyme/PBS, with/without irradiation, n = 4). (E) Fluorescence images of [Ru(dpp)3]Cl2 in differently treated 4T1 cells (ISSzyme/PBS, hypoxia/ normoxia, n = 4). (F) Invasive ability assay of 4T1 tumor cells. (G) Quantification of [Ru(dpp)3]Cl2 in 4T1 cells after different treatments. (H) Quantification of relative metastatic number of 4T1 cells after different treatments. (I) Quantification of apoptosis/necrosis by flow cytometry in 4T1 cells with different treatments. (J) Flow cytometry apoptosis/necrosis analysis of differently treated 4T1 cells based on Annexin V-APC/PI staining assay.

04

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In vivo experiments on mice

In vivo experiments validated the photoacoustic imaging capability of ISSzyme. Two 4T1 xenograft tumors were implanted in the back of BALB/c mice, which were injected with ISSzyme and PBS as controls. PA images were recorded at different time points using the PACT system, and the results showed that the signals of ISSzyme-treated tumors increased significantly with time, while the PBS control showed almost no change. Quantitative analysis showed that the PA signal of ISSzyme-treated tumors was significantly higher than that of the PBS control. In further experiments, ISSzyme was administered by systemic administration and the subcutaneous 4T1 tumor model was imaged under PACT, which showed that the PA amplitude at the tumor site reached a maximum at approximately 10 hours after injection, and the increase in PA signal was attributed to the accumulation of ISSzyme in the tumor. The results of the in vivo fluorescence experiments were consistent with the photoacoustic imaging results, further demonstrating that ISSzyme reacts with GSH to produce detectable PA signal changes at the tumor site, providing an effective method for highly sensitive and specific tumor imaging. In summary, ISSzyme was able to effectively generate PA signal changes at the tumor site, demonstrated excellent targeting and specificity, and provided new possibilities for tumor imaging.

Figure 4.

ISSzyme-assisted tumor PAI (A) Typical PA images of 4T1 tumor-bearing mice at 0.5, 2, 4, and 6 hours after subcutaneous injection of PBS and ISSzyme. (B) Data plot of quantitative PA700/PA990 results as a function of time after injection of PBS or ISSzyme (n = 3). (C) Typical PA images of 4T1 tumor-bearing mice before and after i.v. injection of ISSzyme at 2, 4, 6, 8, 10, 12, and 24 hours. (D) Quantitative histogram of PA700/PA990 after ISSzyme injection (n = 3). (E) Horizontal and vertical maximum intensity projections of representative in vivo PAMe imaging (700 nm) results of 4T1 tumor-bearing mice before and after ISSzyme injection; white dashed line: tumor, red dashed line: ISSzyme.(F) PA700/PA990 quantification of tumor sites before and after systemic administration of ISSzyme. (G) Spatial distribution of ISSzyme in the tumor as observed by PAMe

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Verification of liver accumulation ability

Through a series of PAI experiments, this study validated the ability of ISSzyme to accumulate in the liver. The accumulation of ISSzyme and PB nanoenzymes in the liver and tumor was measured in a 4T1 subcutaneous tumor model. Ten hours after administration, cross-sectional images of the liver and tumor at different wavelengths were recorded using the PACT technique. The results showed that ISSzyme and PB nanoenzymes accumulated in both tumors and liver, and the signal changes induced by ISSzyme were similar to those of PB nanoenzymes. Quantitative analysis showed that PA signals were significantly enhanced in ISSzyme and PB nanoenzymes-treated tumors compared to controls. Specifically, the PA signal of ISSzyme-treated tumors was 2.07 times higher than that of the control group, while the PA signal of PB nano-enzymes-treated tumors was 1.44 times higher than that of the control group. At the liver site, the accumulation of PB nano-enzymes resulted in increased light attenuation, whereas the abundance of liver features in the ISSzyme-treated group was comparable to that of the control group. Further PACT imaging experiments demonstrated that ISSzyme was able to effectively distinguish between tumor and healthy liver tissues. After treatment, the PA signals of ISSzyme-treated tumors were significantly increased, whereas the PA signals of PB nanozyme-treated tumors did not change significantly. These results confirmed the ability of ISSzyme to accumulate in the liver and its ability to differentiate between tumor and healthy tissues. In summary, the accumulation ability of ISSzyme in the liver and its ability to differentiate between tumor and healthy tissues were verified in PAI experiments, which lays the foundation for its further study in preclinical and clinical applications.

Figure 5.

Liver accumulation properties of ISSzyme (A) Typical PA images of mice 10 hours after subcutaneous injection of PBS, PB nanoenzymes, and ISSzyme, demonstrating 4T1 tumor and liver cross sections (n = 3). (B) Maximum intensity projection of PAMe imaging of the right liver lobe of a mouse 10 hours after injection of PBS (left column) and PB nano-enzyme/ISSzyme (right column). (C) Histogram of PA700/PA990 quantitative data for subcutaneous 4T1 tumor and liver cross sections after systemic administration. (D) Histogram of quantitative data of PA tumor/PA liver after systemic administration. (E) Histogram of quantification data of relative PA signaling in the right liver lobe of mice. (F) Representative PA images of mouse liver cross-section (green dashed line) and 4T1 tumor liver metastasis (white dashed line) at 10 hours after injection of PB nanozyme or ISSzyme. (G) PAMe imaging results of the right liver lobe and 4T1 tumor liver metastasis (dashed line) in mice 10 h after injection of PBS, PB nano-enzyme and ISSzyme. (H and I) Histograms of PA700/PA990 quantitative data of 4T1 tumor liver metastasis (H) and liver (I) before and after drug administration. (J) Histogram of PA tumor/PA liver quantification data for 4T1 tumor liver metastasis and liver before and after drug administration.

06

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Verification of tumor hypoxia remission

To assess the efficiency of ISSzyme in alleviating tumor hypoxia, the study examined the expression level of HIF-1a in 4T1 tumor-bearing mice. Animals treated with ISSzyme showed lower HIF-1a red fluorescence intensity compared to the control and laser-treated groups, indicating that ISSzyme was effective in alleviating hypoxia (see Figure 6D). This was further confirmed by quantitative analysis, where HIF-1a expression in the ISSzyme combined with laser treatment group was approximately only 12.17% of that in the control group (Fig. 6C), suggesting that ISSzyme treatment facilitated the decomposition of H2O2 by the on-site-synthesized PB nano-enzymes, and that the released O2 contributed to the amelioration of tumor hypoxia. Lung metastasis of 4T1 tumors was evaluated on day 11 after treatment to determine the effect of metastasis inhibition (Figure 6E). Metastatic tumors were prevalent in the lungs of the control and laser-treated groups, whereas the ISSzyme-treated group exhibited few or no signs of metastasis in the lungs. In addition, when metastatic lesions were observed by H&E staining, cellular staining of metastatic foci was evident in the control and laser groups, while metastatic foci were difficult to be observed in the ISSzyme-treated group. This study monitored blood oxygen saturation (SO2) before and after tumor treatment using multispectral 3D PAMe technology. The analysis showed that the on-site synthesized PB nanoenzymes significantly improved the hypoxic condition (Figure 6F). In conclusion, ISSzyme can effectively inhibit lung metastasis of breast cancer, and its effect is attributed to the alleviating effect of hypoxia by the on-site synthesized PB nanoenzymes.

图6
Schematic illustration of in vivo treatment and tumor hypoxia remission (A and B) Tumor growth curves (A) and body weights (B) of different groups after different treatments. (C) Relative HIF-α expression after different treatments. (D) Light microscopic pathological analysis of resected tumor sections after different treatments, including hematoxylin-eosin staining, TUNEL test, Ki-67 staining and HIF-α staining. (E) Pictures of lung metastases in different treatment groups and H&E-stained lung sections. (F) 3D PAMe images of SO2 collected at 0 h and 10 h after receiving ISSzyme treatment.

07

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Summary

By comparing with conventional PB nanoenzymes, this study verified the feasibility of on-site synthesis of nanoenzymes. The novel nano-enzyme ISSzyme not only retains the tumor therapeutic and hypoxia-relieving effects of conventional PB nano-enzymes, but also minimizes accumulation in metabolic organs such as the liver.ISSzyme is capable of responding to GSH in the tumor microenvironment to enable photoacoustic imaging-guided photothermal therapy (PAI-PTT), while reducing damage to normal tissues during the treatment process.ISSzyme is an innovative nanomedicine for cancer therapy and tumor hypoxia relief. When ISSzyme reacts with GSH, which is highly expressed in tumors, it synthesizes PB nano-enzymes in situ in vivo. This endogenous PB nano-enzyme has peroxidase-like activity, which can decompose H2O2 to produce O2, alleviate tumor hypoxia and inhibit metastasis.The light-absorbing properties of PB nano-enzymes towards NIR make ISSzyme an ideal drug for PA-guided PTT.ISSzyme's high sensitivity and specificity towards GSH helps to minimize the false-positive image signals and therapeutic side effects. Its "no liver accumulation" property not only reduces damage to the liver and other metabolic organs, but also helps to identify tumors from a strong endogenous background. ISSzyme has demonstrated high biocompatibility in both in vitro and in vivo evaluations. In summary, the on-site synthesis of nano-enzymes by ISSzyme provides new ideas and insights for the design and application of artificial enzymes in the future.

DOI：10.1016/j.isci.2023.106066.

source (of information etc)：iScience.
2023 Feb 17; 26(2): 106066.

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