A Multifunctional Biodegradable Prussian blue Analogue for Synergetic Photothermal/Photodynamic/Chemodynamic therapy and Intrinsic Tumor Metastasis Inhibition


 Background

To date, various Prussian blue analogues (PBA) have been prepared for biomedical applications due to their unique structural advantages. However, the safety and effectiveness of tumor treatment still need further exploration.
Results

This contribution reports a facile synthesis of novel PBA with superior tumor synergetic therapy effects and a detailed mechanistic evaluation of their intrinsic tumor metastasis inhibition activity. The as-synthesized PBA have a uniform cube structure with a diameter of approximately 220 nm and showed high near infrared light (NIR) photoreactivity, photothermal conversion efficiency (41.44%) and photodynamic effect. Additionally, PBA could lead to chemodynamic effect which caused by Fenton reaction and ferroptosis. The combined therapy strategy of PBA exhibit notable tumor ablation properties due to photothermal therapy (PTT)/photodynamic therapy (PDT)/ chemodynamic therapy (CDT) effect without obvious toxicity in vivo. The PBA also demonstrate potential as a contrast agent for magnetic resonance imaging (MRI) and photoacoustic (PA) imaging. More importantly, careful investigations reveal that PBA displays excellent biodegradation and anti-metastasis properties. Further exploration of this PBA implies that its underlying mechanism of intrinsic tumor metastasis inhibition activity can be attributed to modulation of epithelial mesenchymal transition (EMT) expression.
Conclusions

The considerable potential exhibits by as-synthesized PBA make it an ideal candidate as a synergetic therapeutic agent for tumor treatment.


Introduction
Recently, nanotechnology as a new strategy for cancer diagnosis and treatment has received much attention in biomedical science. [1][2][3] PTT, a cancer treatment developed from the application of nanotechnology, is based on the conversion of NIR radiation to induce hyperthermia and then "burn" cancer cells. [4][5][6][7][8][9] Due to its convenient operation, minimal side effects and noninvasivity, PTT has been considered an alternative approach for clinical usage. [10] However, the therapeutic effect is limited by the use of a single treatment at a safe dosage, uneven distribution of photothermal agents and endothermic effect of blood vessels around tumors also affect the ablation e cacy. [11] Therefore, the combined therapy strategies, such as PTT/PDT, PTT/CDT and PDT/CDT have been proposed and achieved good performance. [12][13][14] PDT is another type of phototherapy for tumors and is characterized by reactive oxygen species (ROS) generation in tumor environment to kill cancer cells. [15] In addition, PDT can also decrease tumor nutrition by destroying nearby blood vessels. [16] Another therapy strategy based on ROS generation is called as CDT, which is depedent on Fenton reaction under ferrous ion or transition metal ion. [17] Since the high H 2 O 2 content (100 µM to 1 mM) in tumor microenvironment, Fenton reaction can transfer H 2 O 2 to toxicant ROS (·OH) and generate O 2 at the same time under ferrous ion catalysis. [13] Thus, CDT process can produce O 2 for PDT while near infrared light irradiation ameliorate ·OH generation in Fenton reaction, which can improve cancer therapeutic e cacy. [17,18] To achieve better PTT/PDT/CDT therapeutic e ciency, the preparation of photothermal agent and photosensitizer (PS) which can trigger Fenton reaction or Fenton-like Reaction is critical. To date, some NIR-responsive materials have been developed for photothermal therapy or combined therapy strategies in tumor treatment. [19][20][21][22] However, potential safety issues for nanomaterial application always exist concomitantly, and the main contradiction is particle toxicity caused by low biodegradability in vivo. [23,24] The high photothermal conversion e ciency of nanomaterials leads to good PTT performance, but because long-term toxicological evaluations are still scarce, the application of nanomaterials for clinical transformation is limited. [10,25] Therefore, it is meaningful to synthesize nanomaterials for PTT/PDT/CDT therapy strategies from old substances that have been approved for clinical applications.
Prussian blue (PB) has been used as an ancient dark blue pigment since 1704, and because it is porous and easy to chelate with heavy metals, it has been developed as a drug named "Radiogardase" and approved by the U.S. Food and Drug Administration (FDA) for internal contamination treatment. [26][27][28] PB has the general formula of [Fe II (CN) 6  metastasis with no effect on cell proliferation capacity. [40] Therefore, it should be meaningful to discover nanomaterials which have excellent biocompatibility and anti-metastasis properties for tumor therapy.
Hence, in this study, a novel PBA nanoparticle is synthesized via a facile protocol under mild conditions. [41] According to previous reports, the prepared PBA is expected to be sensitive to NIR radiation and could be an outstanding candidate for PTT/PDT/CDT strategies. Excitingly, at least in nanomaterial applications, the anti-metastatic properties and biodegradation potential of this PBA for tumor therapy are unique (Scheme 1).

Synthesis and characterization of PBA
The synthesis of the PBA was based on a precipitation method with slight modi cation, [41] and compared with the reported studies, all the required materials and steps in this study were simpler and more facile. [2,42] SEM and TEM images showed that the prepared PBA has a uniform cube structure with a diameter of approximately 220 nm ( Fig. 1A-B). Moreover, Fig. 1B also showed that PBA nanoparticles were hollow and could thus be used as drug carriers. Figure 1C displayed a clear lattice structure of PBA recorded by TEM, which demonstrated that PBA had good crystallinity. Additionally, the crystalline structure of PBA was further detected by XRD, and the diffraction pattern con rmed the formula of pure Co 3 [Fe(CN) 6 ] 2 ·10H 2 O (JCPDS 86-0502) (Fig. 1D) produced after preparation.

Imaging function of PBA
Iron-containing nanoparticles have been extensively studied as potential contrast agents for MRI. [43,44] It has been reported that PB has extraordinary performance as an MRI contrast agent due to its special structure. [7,28,45] Nanostructures of PB contain low-spin Fe 2+ -C (S = 0) and high-spin Fe 3+ -N (S = 5/2), which could shorten the time of transverse and longitudinal relaxation on water protons. In addition, each Fe 2+ -C ≡ N-Fe 3+ unit of PB has ve unpaired electrons that can form coordination bonds between water molecules and Fe 3+ , causing relaxation inside PB and enhancing the contrast effect. [7] In this study, the synthesized PBA was considered to have a similar structure and character to PB, so this PBA was also expected to have the potential to be an MRI contrast agent. Figure 1E demonstrated that different concentrations of PBA displayed various degrees of T 2 -weighted contrast property due to the Fe 3+ superparamagnetic centers, and r 2 relaxation value was determined to be 61.381 mM − 1 s − 1 , implying that PBA is a prospective contrast agent for MRI.
PA is a new visual imaging technology based on light absorption-induced ultrasonic waves. [46][47][48][49] Because of the excellent performance on NIR region absorption, the PBA could also be used as a contrast agent for PA. The results are shown in Fig. 1F. As the PBA concentrations increased, the PA signal intensity was also enhanced and presented an almost linear increase. These results demonstrated that the PBA can be performed as a contrast agent for PA imaging.
2.3 Photothermal, photodynamic and chemodynamic effect of PBA As shown in Fig. 2A, 1 mg mL − 1 PBA presented a brownish black color and good dispersed in water. The UV-vis absorption spectrum showed that PBA had broad and strong absorption in NIR region (500-900 nm), which demonstrated that PBA had potential application as an NIR-driven photothermal agent. Subsequently, the photothermal effect of PBA was detected by temperature change of different concentrations of PBA under 808 nm irradiation for 10 min with PBS solution as a control. The temperature of PBA solution rapidly increased (7-40°C) with increasing time and concentrations (0.1-2 mg mL − 1 ), while control group had a negligible temperature change (3°C) (Fig. 2B). Moreover, the data showed that more than 0.5 mg mL − 1 PBA can rapidly heat to hyperthermic conditions (> 45°C), which further demonstrated the photothermal ablation potential of PBA for cancer cells. [50] In addition, we also investigated the temperature changes of 1 mg mL − 1 PBA with irradiation powers varying from 0.1 to 2 W cm − 2 ; the results showed a strong dependence on irradiation energy (Fig. 2C), and 2 W cm − 2 irradiation was adopted in subsequent experiments. The photothermal stability of PBA was further studied by laser on/off experiments for ve cycles. The results were presented in Fig. 2D, the temperature changes of PBA were similar in ve cycles, which indicated the excellent photothermal stability of PBA. According to these data, the photothermal conversion e ciency of PBA was calculated, and the η value was approximately 41.44%, which demonstrated the excellent photothermal conversion performance of PBA.
The ·OH production was measured by MB and OPD probe. The diminish of MB indicates Fenton reaction occurred and diaminophenazine (DAP) can generate by OPD under Fenton reaction, both of these tests can detected at speci c absorbance. As the results showed in Fig. 2E and ·OH, and more O 2 lead to more PDT effect.

In vitro combined therapy effect of PBA
Because of the excellent performance of PBA for NIR response, the photoablation property in vitro was investigated in 4T1 cells by LIVE/DEAD™ staining. As shown in Fig. 3A, the living cells were dyed with FITC, exhibiting green uorescence, while the dead cells presented red uorescence due to Texas Red staining. After PBA + NIR treatment, signi cant red uorescence was observed under a uorescence microscope, which demonstrated massive cell death. In contrast, PBA alone group exhibited green uorescence caused by living cells, which was similar to PBS and PBS + NIR treatment groups.
Surprisingly, in addition to the photothermal property, the photodynamic and chemodynamic effect of the PBA was also observed in 4T1 cells through DCFH-DA staining. DCFH-DA is a commonly used dye to detect ROS based on oxidation reactions, and the generation of ROS will be marked by intense green uorescence. Figure 3B  Ferroptosis is one of cell death pathway which dependent on iron and ROS accumulation. Since Fe 2+ released and ROS generated capacity of PBA was con rmed, it was considerable that the underlying mechanism was related with ferroptosis. Glutathione peroxidase (GPX4) as a critical enzyme in ferroptosis process, its activity can prevent the toxicity of lipid peroxide. The less content of GPX4 causes more serious ferroptosis of cell death. [53] Nuclear factor E2-related factor 2 (NRF2) is also an antiferroptosis factor. After PBA treatment, both of GPX4 and NRF2 were declined in 4T1 cells, indicated ferroptosis occurred ( Fig. 3C-D). Additionally, iron metabolism pathway plays an important role in ferroptosis process. PBA could release lots of Fe 2+ in tumor microenvironment which supplied more resource of Fe 2+ for tumor cells and further disturbed iron metabolism. The results showed transferrin receptor 1 (TFR1), ferritin heavy chain 1 (FTH1) and ferritin light chain (FTL) were all increased after PBA treatment which demonstrated disorders of iron metabolism (Fig. 3E-G). Thus, PBA induced ferroptosis was caused by combined effect of down regulated GPX4, NRF2 and increased TFR1, FTH1, FTL, as well as Fenton reaction (Fig. 3H).

Combined treatment of PBA in vivo
To further con rm combined therapeutic effect of PBA in vivo, a tumor-bearing mouse model was established. Figure 4A presented the comparison of temperature change between PBS and PBA treatment with laser irradiation for 10 min. It was clear that PBA treatment could heat the tumor site above 45°C after 1 min of irradiation, while the control group remained at 37°C. More details were recorded in Fig. 4B. The temperature of PBA group dramatically rise to 55°C in 200 s irradiation and then increased slowly, and after treated for 400 s, the temperature of tumor site was maintained at approximately 60°C. In contrast, the temperature of PBS group increased slowly during irradiation and nally remained at approximately 45°C.
According to the above results, the long-term combination therapeutic effect on PTT/PDT/CDT treatment of PBA was investigated in vivo. Tumor-bearing BALB/c mice were randomly divided into four groups: PBS, PBA, PBS + NIR and PBA + NIR. Figure 4C displayed the weight changes of mice in different groups, and no obvious difference was observed during whole process. Tumor volumes were recorded as V/V 0 in Fig. 4D. It was clearly demonstrated that after PBA injection and laser irradiation, the tumor was digested and cleaned up; more importantly, there was no tumor recurrence during the 27-day observation period. In contrast, the other three groups showed the same tumor growth rate, and nal tumor size is intuitively presented in Fig. 4E. Compared to small recovered scab of PBA + NIR group tumor, the others were extremely huge. Figure 4F

Evaluation of PBA on tumor metastasis
The previous studies demonstrated that although some nanomaterials had a small impact on cancer cell proliferation, they impair the migration and metastasis of tumors. [40,[54][55][56] Hence, we also explored the effect of PBA on tumor metastasis in vivo and in vitro. After a 27-day observation experiment, the lungs of each mouse were harvested, and metastatic nodes were counted. The tumor metastatic nodes were clearly observed on the surface of lung (Fig. 5A), and a typical histopathological slice was presented by H&E staining (Fig. 5C). After PBS, PBA and PBS + NIR treatment, some dark dyed nodules with clear edges were observed in the lung tissue, which further con rmed the tumor metastasis. In addition, statistical data of metastatic node numbers in PBS-and PBA-treated groups were signi cantly different, which suggested the potential utility of this PBA as a synergistic anti-metastasis agent to suppress tumor invasion (Fig. 5B).
Further mechanistic exploration of PBA on tumor metastasis was also studied in 4T1 cells in vitro. The transwell experiment was used to con rm the effect of PBA on cell invasion. After treatment with 0, 10 and 50 µg mL − 1 PBA for 24 h, the intensity of migrated cells was reduced after PBA treatment (Fig. 5D-E).
These results combined with those in Fig. 4 showed that PBA had no signi cant effect on cytotoxicity without laser irradiation, which demonstrated that reduction of invasive cells in Fig. 5D was contributed by t tumor metastasis-preventing property of PBA.
EMT is a typical biological phenomenon in tumor metastasis and is also an important target for tumor therapeutic approaches. EMT is characterized by a decrease in cell adhesion and the expression of proteases such as matrix metalloproteinases (MMPs). [57] Epithelial cadherin, also called E-cadherin, is a tight junction molecule between cells, and the decomposition of E-cadherin induces tumor metastasis.
[58] As shown in Fig. 5F-H, the expression of E-cadherin (CDH-1) was increased after PBA treatment, while that of MMP9 was decreased at the same time, which agreed with the results of the transwell experiment.
To summarize, PBA exhibited surprising potential for inhibition of tumor metastasis, which was con rmed by in vitro and in vivo experiments. The mechanism of decreased tumor invasion by PBA was achieved by suppressing MMP9 expression and recovering E-cadherin function.

Biodegradability and biocompatibility of PBA
Since more and more attention has been paid to application of nanomaterials for cancer diagnosis and treatment, safety concerns also appear at the same time. The most important issues are cytotoxicity, long-term bioaccumulation and nanoparticle aggregations caused vascular blockage. [24] Therefore, it is very meaningful to nd materials with good biodegradability for use in body. [59,60] Due to the biocompatibility and FDA approval of PB, this traditional dyestuff has been considered as a safe agent for PTT treatment of tumors. However, the long-term toxicity and physical aggregation of PB are still to be solved. [7] To evaluate the same problem in PBA, 1 mg mL − 1 PBA was prepared in different solvents (distilled water, PBS, 10% FBS, 10% BSA and EDTA), among which 10% FBS and 10% BSA were used to simulate the internal environment. The results were recorded in Fig. 6A, and the images from day 0 to day 12 were displayed. The different tubes of prepared solutions on day 0 appeared to be uniform brownish black except for the pink color of EDTA tube (possibly due to chelation with Co 2+ in PBA). After 3 days, the color of 10% FBS and 10% BSA groups gradually faded, especially on day 12; these two groups basically recovered to original solvent color, while the color of the others had no obvious change. These results suggested that the synthesized PBA may decompose gradually internally, but in vivo experiments are still needed to determine degradation effect and mechanism.
Besides that, the biocompatibility of PBA was evaluated by hemolysis test and results revealed that even 1 mg mL − 1 PBA could remain the normal condition of erythrocyte compared positive control group (Figs. 6B). The biocompatibility of PBA was also investigated during cancer treatment, the major organs (heart, liver, spleen and kidney) were collected to weigh and stain with H&E. The nal histograms and images are displayed in Fig. 6C-F and Fig. 7. Both the results showed that these organs had no noticeable pathological change during the 27-day observation, which demonstrated that no organ damage occurred after PBA treatment except in spleen. The abnormal performance of spleen could be induced by tumor development, which caused immune response in the whole body under PBS, PBA and PBS + NIR treatment.

Conclusions
In this study, a novel NIR photoreactive material, a PBA, was prepared via a facile protocol. The broad NIR absorption gave PBA potential for use in photothermal and photodynamic therapy for tumor treatment. Meanwhile, PBA also had ability for chemodynamic therapy due to Fenton reaction caused by ferrous ion and the underlying pathway was related to ferroptosis. Additionally, the PBA also exhibited excellent potential as MRI and PA contrast agents. More importantly, the outstanding biodegradation and antimetastatic properties of PBA encouraged its use as a unique nanoparticle for cancer treatment. Further exploration revealed that the underlying mechanism by which PBA suppressed cancer invasion was by regulating EMT protein expression.

Characterization of PBA
Scanning electron microscopy (SEM) (S-4800 FESEM, Hitachi, Japan) was used to investigate the surface of PBA. More information about the morphology and internal structure of PBA was provided by transmission electron microscopy (TEM) (JEM 2100F, JEOL, USA) and energy-dispersive X-ray spectroscopy (EDS) was presented by a Thermol UltraDry EDS system attached to the TEM instrument. The crystal structure of PBA was characterized by X-ray diffractometry (XRD) (X'Pert Pro MPD, PANalytical, Netherlands). The absorption spectrum was recorded by a UV-vis spectrophotometer (UV-1800, Shimadzu, China). MRI was obtained by a 3.0 T MR imaging instrument. Photoacoustic tomography (PAT) was collected on a multispectral optoacoustic tomography scanner (MSOT, iThera Medical, Germany).

Photothermalperformance
To investigate the photothermal properties of PBA, different concentrations of PBA (0.1 0.2, 0.5, 1, 2 mg mL -1 ) were prepared and treated with 808 nm irradiation at various powers (0.1, 0.5, 1, 2 W cm -2 ) for 6 min. The real-time temperature of sample was measured by an FLIR thermal camera. A laser on/off experiment was used to estimate the photothermal stability of PBA. The whole process included ve continuous cycles, each of which was under 808 nm irradiation at 2 W cm -2 for 600 s in the laser on condition, and then turned the laser off until the solution had cooled down to room temperature naturally. Furthermore, the photothermal conversion e ciency (η) of PBA was calculated. [12] T max : maximum equilibrium temperature for PBA solution

In vitro photothermal property
First, 4T1 cells were seeded into 96-well plates overnight and divided into four groups: PBS, PBS+NIR, PBA and PBA+NIR groups. Then, the culture media were removed by rinsing with PBS three times, and the samples were treated as follows: the PBA and PBA+NIR groups were treated with culture medium which contained 1 mg mL -1 PBA, and the PBS and NIR groups were treated with the same dosage of culture medium contained PBS. After incubated for 4 h, the media were removed, and the samples were rinsed with PBS three times, and then the PBS+NIR and PBA+NIR groups were accepted 808 nm irradiation at 2 W cm -2 for 10 min. Last, all of these groups were dyed with the LIVE/DEAD TM Cell Imaging Kit which the green uorescence exhibited living cells, while dead cells were marked red.

Intracellular reactive oxygen species (ROS) generation
The pretreatment process with 4T1 cells was similar to the LIVE/DEAD TM Cell Imaging Kit experiment. Then, the ROS Assay Kit was used to detect intracellular ROS generation. The DCFH-DA probe was diluted in FBS-free DMEM (10 μmol L -1 ) and replaced with culture medium. After culturing for another 20 min, all groups were washed with FBS-free DMEM three times, and then NIR groups were treated with 808 nm irradiation at 2 W cm -2 for 10 min. All groups were recorded by uorescence microscopy.

Evaluation of the photothermal effect in vivo
When the tumor-bearing mouse models were successfully established, they were divided into PBS+NIR and PBA+NIR groups randomly, and 50 μL PBS or PBA (10 mg mL -1 ) was injected into the tumor site.
After that, the mice were exposed under 808 nm irradiation at 1 W cm -2 for 10 min. The real-time temperature change and images of the whole mouse body were recorded on an FLIR thermal camera.

Animal experiment
The tumor-bearing BALB/c mice were divided into four groups: PBS, PBS+NIR, PBA and PBA+NIR groups. Then, 1 mg mL -1 PBA was prepared in the different solvents and placed at room temperature.

Statistical analysis
All the data in this report were collected and analyzed by IBM SPSS Statistics 20.0, and the quantitative data were organized as the mean ± standard deviation (SD). One-way ANOVA was used to investigate the comparison among groups. P<0.05 was considered a signi cant difference.

Declarations
Ethics approval and consent to participate All animal studies followed the guidelines approved by the ethics committee of Guangdong Medical Laboratory Animal Center (B202009-1).
Consent for publication