Bufotalin-loaded biomimetic prussian blue nanoparticles for colorectal cancer chemo-photothermal therapy via induce ferroptosis

doi:10.21203/rs.3.rs-2580869/v1

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Bufotalin-loaded biomimetic prussian blue nanoparticles for colorectal cancer chemo-photothermal therapy via induce ferroptosis

https://doi.org/10.21203/rs.3.rs-2580869/v1

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Background: As a ferroptosis-inducing platform, based on its release of iron and ferrous ions to induce production of reactive oxygen species (ROS), it has attracted extensive attention in cancer treatment. Since iron-based nanomaterials such as prussian blue (PB), can only produce limited ROS. Bufotalin (CS-5), a promising ferroptosis inducer, was introduced to improve the efficiency of ferroptosis. However, the clinical application of CS-5 was limited by cardiotoxicity and high renal clearance rate. Therefore, we constructed a biomimetic nanosystem (PB@CS-5@M) with satisfactory tumor enrichment ability and long blood circulation time for the treatment of colorectal cancer induced by ferroptosis.

Results: In this work, the PB@CS-5@M has efficient loading of CS-5, which significantly reduces the cardiotoxicity of CS-5. After photothermal therapy, the high temperature caused by laser stimulation triggers the release of CS-5, iron and ferrous ions to kill colorectal cancer cells. Furthermore, the ability of CS-5 to significantly down-regulate the expression of GPX4 and HIF-1a, induce ROS production and damage mitochondria to induce ferroptosis in colorectal cancer was amplified. In addition, the down-regulation of HSP70 expression under laser stimulation effectively improved the chemosensitivity of HCT116 cells to CS-5.

Conclusions: The chemotherapeutic effect of CS-5 combined with the photothermal effect of PB can effectively treat colon cancer by inducing ferroptosis.

Prussian blue

Bufotalin

Ferroptosis

Chemo-photothermal therapy

Colorectal cancer

Colorectal cancer (CRC) is the word’ s third leading cancer and the second leading cause of cancer related deaths worldwide [1-3]. The treatment of colorectal cancer includes surgery, radiotherapy, chemotherapy, targeted therapy, and ablation. Although chemotherapy has made progress, the cure rate is far behind that of breast cancer and lung cancer considering the liver and lung metastasis of colorectal cancer[3-6]. In addition, chemotherapy may result in serious adverse effects, such as hematotoxicity and drug resistance, leading to poor prognosis for patients[7-9]. In view of this, there is an urging need for a new treatment for colorectal cancer.

It is reported that Bufotalin(CS-5) is a natural compound derived from Chansu[10], a traditional Chinese medicine, which can effectively treat melanoma[11], lung cancer[12], and liver cancer[13]. However, the strong cardiotoxicity of CS-5 limits its clinical application[10]. Zhao et al. demonstrated that the mechanism of bufotalin killing tumor cells is to induce G2/M phase cell cycle arrest and apoptosis, thereby inhibiting A375 cell proliferation[14]. Zhang et al. also proved that bufotalin inhibits GPX4 expression, and promotes the production of ROS and the level of intracellular iron (Fe2+ form), thereby inducing ferroptosis and inhibiting the proliferation of A549 cells, which suggested the prospect of developing CS-5 as a novel ferroptosis inducer for antitumor therapy[15].

Ferroptosis is an iron-dependent mode of cell death triggered by the inactivation of glutathione (GSH)-dependent antioxidant defenses, leading to the accumulation of toxic lipid ROS, which differs from other forms of cell death[16]. Its characteristics include increased ROS level, decreased glutathione peroxidase (GPX4), and damaged mitochondrial[17, 18]. In the latest study, the combination of siramesine and lapatinib can induce ferroptosis, which may be a solution for lapatinib resistance to breast cancer therapy[19]. However, due to the high renal clearance and difficulty in accurately reaching the tumor sites, the clinical application prospect of many ferroptosis-inducing drugs was limited [20]. It deserves mentioning that many nanomaterials were prepared to overcome these limitations. Due to their incomparable advantages, including non-toxic, longer blood circulation time, tumor homing ability high drug loading rate and controlled drug release, they provide a promise for specific ferroptosis cancer therapy [21-23]. ]Iron-based nanomaterials have gained advantages in inducing ferroptosis of cancer cell, due to its superiority of releasing iron and ferrous ions to induce ROS generation[20, 24].

Prussian blue (PB), as an iron-based nanomaterial, possesses the advantages of high drug loading rate and excellent photothermal properties, more importantly, the U.S. Food and Drug Administration (FDA) has approved prussian blue for the treatment of radiation diseases and MRI, because of its good biological safty[25, 26]. Chen et al. constructed a triple therapy based on curcumin and 5-Fu loaded with prussian blue, and it has an excellent anti-colorectal cancer effect[27]. Temperature is the main factor for ROS production, research shows that PB with photothermal therapy (PTT) can release iron and ferrous ions to promote ROS production [28, 29], which is expected to further improve the tumor cell ferroptosis in tumor treatment. Recently, there were no studies have confirmed the potential of developing CS-5 for tumor cell ferroptosis for colorectal cancer treatment. Prussian blue for photothermal therapy has the potential to induce ferroptosis. Therefore, a biomimetic nanosystem (PB@CS-5@M) with NIR laser response was constructed to combine CS-5 with prussian blue for chemo-photothermal therapy against colorectal cancer by inducing ferroptosis. PB@CS-5 (PC) was prepared by loading chemotherapy drug CS-5 with the prussian blue (PB), and then PB@CS-5@M (PCM) was further prepared by combining a hybridized erythrocyte-tumor membrane (M) with PB@CS-5. The hybridized erythrocyte-tumor membrane endows PB@CS-5@M with tumor-targeting properties and longer circulation time. CD47 molecules on the surface of erythrocyte membrane avoided immunogenic clearance, the half-life of nano-drug in blood circulation is prolonged, and the tumor cell membrane is targeted in the same type. It is widely reported in nanomedicine that the best therapeutic effect can be achieved by using mixed membranes[30, 31]. Xiao et al. successfully constructed a nano drug system for the treatment of cervical cancer, which use a hybrid membrane to wrap with Prussian blue nanoparticles of CS-6 and IND[32].

In this work, PB@CS-5@M was designed to attenuate the cardiotoxicity of CS-5 and assist CS-5 to achieve efficient accumulation and release at the tumor site under tumor microenvironment. CS-5 down-regulates the expression of GPX4, reduces mitochondrial membrane potential and increases ROS, thus inducing ferroptosis. PB directly killed tumor cells under laser stimulation, and released iron and ferrous ions to boost ROS generation and ferroptosis, which realized double-hit ferroptosis treatment for colorectal cancer. Moreover, the nanocomplexes down-regulates the expression of HSP70 under laser stimulation to enhance the chemotherapy sensitivity of HCT116 cells to CS-5.

2.1. Materials

K₃[Fe(CN)₆]·3H₂O was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd (China). Poly (N-vinylpyrrolidone) (PVP) was purchased from Zhanyun Chemical Co., Ltd. (China). Bufotalin (CS-5) was acquired from Dalian Medical University. Rhodamine was obtained from Sigma-Aldrich (USA). JC-1 kit and DCFH-DA probe were purchased from Beyotime Biotechnology (China). MTT and DAPI were purchased from Solarbio Technology. Calcein AM/PI kit was obtained from Yeasen Biotechnology (China). HE staining solution purchased from biosharp Technology Co., Ltd (China).

2.2. Cell lines and animals

HCT116, NIH-3T3, and SW480 cells were purchased from Dalian Meilun Biotechnology Co., Ltd (China). Human vascular smooth muscle cells (VSMC) and RAW 264.7 cells were obtained from Xiangya Central Laboratory (Hunan, Changsha, China). Female nude BALB/c mice (4-5 weeks old) were obtained from SJA Laboratory Animal Co., Ltd (Hunan, China).

2.3. Synthesis of Prussian blue nanoparticles (PBNPs)

PBNP was synthesized according to the previous method[33]. 7.5 g PVP and 330mg K₃[Fe(CN)₆]·3H₂O were weighted and dissolved in 0.1 M 100 mL HCl, and stirred at 800 rpm for 1 h, and then conduct oil bath at 80 °C for 20 h. PBNPs were collected after centrifugation, washing, and freeze-drying.

2.4. Preparation of PB@CS-5@M (PCM)

Preparation of RBC membrane (RBCm): RBC cell membranes were prepared as previously reported[34]. Briefly, the fresh blood collected from healthy mice was washed with PBS (pH 7.4) for 5 times. The red blood cell layer was collected and mixed with 25% PBS (pH 7.4) and lysed on ice for 2 h. Subsequently, the cracked red blood cells were centrifuged at 12000 g and washed until the supernatant was colorless, then stored at -80 ℃ until for use.

Preparation of HCT116 membrane (HCTm): HCT116 cell membranes were prepared according to instructions of membrane protein extraction kit (Beyotime Biotechnology, China)[35]. HCT116 cells were scraped down and washed with PBS (pH 7.4), then the cells were centrifuged at 800 g, and the precipitate was collected and resuspended in reagent A. Next, after five repeated freeze-thaw cycles, the samples were centrifuged at 800 g, the supernatant was collected and centrifuged at 12000 g, and the precipitation of cell membranes was collected and stored at -80 ℃ until for use.

Preparation of RBC-HCT hybrid membrane: After ultrasonication of RBCm and HCTm respectively, it was filtered with a 0.22 μm filter membrane, and then mixed at a ratio of 1:1.

Preparation of PCM: 950 μL of PB (1 mg/mL) and 50 μL of CS-5 (1 mg/mL) were mixed and stirred at 800 rpm for 12 h, then the mixture was centrifuged at 13500 g for 15 min and the precipitate of PC was collected. 1 mL PC (1 mg/mL) and 1 mL RBC-HCTm (1 mg/mL) were mixed and stirred at 37 ℃ for 2.5 h, then then the mixture was centrifuged and PB@CS-5@M was acquired.

2.5. Characterization

TEM was performed to capture the image of PB and PCM using JEM-2100 microscope (JEOL, 200 kV). The size and zeta potential of PB, PC, and PCM were determined by Zetasizer Nano ZS (Malvern, UK). The UV–vis spectrophotometer (Beckman Coulter Inc., USA) was conducted to detect the characteristic absorption peaks of PB, CS-5, PC, and PCM. RBC-HCT116 membrane fusion was characterized by laser confocal microscope (Olympus FV1000, Japan). Membrane proteins including RBCm, HCTm, RBC-HCTm, and PCM were determined by SDS-PAGE.

2.6. Photothermal performance characterization

Temperature change curve and infrared thermal images of PBS, PB, PC, and PCM (0.2 mg/mL) after 808 nm laser irradiation (1.2 W/cm²，5 min) were recorded by the infrared camera (FLIRC2). The temperature change curve of PCM at different concentrations was also measured to evaluate the temperature rising capability. Photothermal curve of 0.5 mL PCM (0.2 mg/mL) were designed to calculate photothermal conversion efficiency. Photostability of PCM was evaluated by laser on/off for 6 cycles, and the characteristic peaks before and after laser irradiation were measured by UV–vis spectrophotometer.

2.7. Evaluation of biocompatibility

The toxicity of PB and PBM to different cells (HCT116 cells, SW480 cells, NIH-3T3 cells, and VSMC cells) was detected by MTT assay. Briefly, when the cell density reached 80% in 96-well plates, different concentrations of PB and PBM (0, 12.5, 25, 50, and 100 µg/mL) were added to the Petri dish and treated for 24 hours, the absorption peak at 490 nm was detected by MTT assay and the cell activity was calculated.

The hemolysis rates of red blood cells in different samples were determined by spectrophotometry. PB, PC, and PCM were added into 4 % suspended red blood cells to the final concentrations of 0, 25, 50, 100, and 200 µg/mL, respectively. In addition, the positive control was treated with ddH₂O, and the negative control was treated with PBS. After incubation at 37 ℃ for 6 h, centrifuged at 3000 g (5 min, 4 ℃), the absorbance of the supernatant at 540 nm was detected with microplate. The hemolysis rate was calculated and erythrocyte morphology was observed under the microscope.

Platelets extracted from fresh whole blood were incubated with PB, PC, and PCM (200 µg/mL) at 37 °C for 4 h. Thrombin treatment group was used as the positive control, and the PBS treatment group was used as the negative control. The absorbance of all samples at 650 nm was detected and the coagulation rate was calculated.

2.8. Cell uptake and immune escape assay

Cell uptake assay: HCT116 cells were seeded into a 12-well plate and cultured. When the cell density reached 60%, 40 µg/mL of rhodamine-labeled PB coated with the hybrid membrane (PB@M^Rho) was added at 0, 2, 4, 6, and 8 h, respectively. The nucleus was stained with DAPI, and the images were captured by laser confocal microscopy.

Immune escape assay: The RAW 264.7 cells (5 × 10⁴) were seeded into a 12-well plate and cultured overnight, and PB^Rho and PB@M^Rho (0, 25, 50, and 100 µg/mL) were added and cultured for 6 h, respectively. The nucleus was stained with DAPI, and the cells were imaged through the laser confocal microscope.

2.9. Antitumor effect and mechanism in vitro

Cell viability: The HCT116 cells were placed into 96-well plate at a density of 8 × 10³ per well and cultured for 24 h, and then treated with PBS, PB, PC, PCM, PB + L, PC + L, and PCM + L (PB: 30 µg/mL, CS-5: 0.0889 µg/mL). The laser was irradiated after 6 h (808 nm, 1.2 W/cm², 5 min), then incubated for 18 h. Cell viability was detected by MTT assay.

Live-death staining: HCT116 cells were seed and cultured in 24-well plates for 24 h, and then treated with PBS, CS-5, PB, PC, and PCM with laser or without laser (PB: 30 µg/mL, CS-5: 0.0889 µg/mL), respectively. The cells were stained with Calcein AM/PI kit and imaged with laser confocal microscope.

ROS detection: When the HCT116 cells confluence reached 60% in 24-well plates, it was treated with PBS, PB, PC, and PCM with laser and without laser (PB: 30 µg/mL, CS-5: 0.0889 µg/mL), respectively. After incubation with the DCFH-DA probe for 30 min, the cells were imaged under a fluorescence microscope.

Detection of Mitochondrial membrane potential (MMP): The HCT116 cells were cultured in 12-well plate, then was treated with PBS, CS-5, PBM + L, PCM, and PCM + L (PB: 30 µg/mL, CS-5: 0.0889 µg/mL), respectively. The laser was irradiated about 6 hours later, and then incubated for 18 h, cells were stained with JC-1 kit for 20 min and then imaged by laser confocal microscope.

Western blotting: When the HCT116 cell confluence were reached 60% in 6-well plates, it was treated with PBS, PB, PB + L, CS-5, PCM, and PCM + L (PB: 30 µg/mL, CS-5: 0.0889 µg/mL), respectively. Cells were washed with PBS and cracked to extract proteins. The expression of C-Caspase-3, P53, Bax, and Bcl-2 was detected to evaluate the level of apoptosis, and the level of ferroptosis was evaluated by measuring the expressions of GPX4 and HIF-1a. In addition, the cells were treated with PBS, PB, PB + L, PBM + L, PC + L, and PCM + L, respectively, and the protein was extracted to evaluate the expression of HSP70.

2.10. Biodistribution and pharmacokinetics in vivo

When the tumor volume of female nude BALB/c mice reached 250 mm³, 100 µL of PB^Ce6 and PB@M^Ce6(The dose of PB was 5 mg/kg) were injected into mice by tail vein injection, respectively. Fluorescence imaging of tumor-bearing BALB/c mice was performed at 2, 4, 6, 8, 12, 24, and 48 h. The mice were dissected after 48 h, and the heart, liver, spleen, lung, kidney, and tumor were collected for imaging.

200 µL of Ce6, PB^Ce6, and PB@M^Ce6(The dose of PB was 5 mg/kg) were injected into tumor-bearing BALB/c mice by tail vein injection. Blood samples were collected at 0, 1, 2, 4, 6, 8, 12, and 24 h after injection. After centrifugation at 3600 g for 5 min, the supernatant was collected for fluorescence imaging and the half-life of different samples was calculated.

2.11. Antitumor effect in vivo

100 μL cells were subcutaneously planted on the back of female nude BALB/c mice at a density of 5 × 10⁶. When the tumor volume reached 100 mm³, the mice were randomly divided into five groups including PBS, PBM + L, CS-5, PCM, and PCM + L (n=5). After the body weight and tumor size were recorded, the mice were treated via the tail vein with 100 μL PBS, PBM, CS-5, and PCM (the dose of PB and CS-5 was 5 mg/kg and 1 mg/kg), respectively, and laser therapy was performed at 12 h later. Significant changes in tumor volume were observed in tumor-bearing mice after eight treatment cycles. The mice were euthanized, the fresh blood of mice was taken for the routine blood test and biochemical test. The heart, liver, spleen, lung, kidney, and tumor were taken for tissue sections, then stained with HE, immunohistochemistry, and TUNEL.

2.12 The analysis of 16S rDNA gene and sequencing

Normal female nude BALB/c mice as the Control group, and female tumor-bearing BALB/c mice were treated with PBS and PCM + L as PBS and PCML groups, respectively (n=5). The feces from three groups of mice were collected for 16S rRNA analysis (lc-bio, Hangzhou, China). All data were analyzed through R software (Version 3.6.3) and the OmicStudio tools at https://www.omicstudio.cn. Correlation calculation was conducted with Pearson and spearman correlation coefficient.

2.12. Statistical analysis

All values are presented as mean ± standard deviations (SD). The statistical difference between the two groups was performed by t-test, and statistical differences above the two groups were performed by One-way ANOVA using GraphPad Prism 8 software (Version 8.4.0). *p < 0.05, **p < 0.01, ***p < 0.001 indicated the difference.

3.1. Characterization of the PCM

The synthesis procedures of PB@CS-5@M are illustrated in Scheme 1. The TEM image of PB shows its stable cube structure and 100 nm nano-particle size with excellent dispersibility (Fig. 1a). After loading with CS-5 and wrapping with the hybrid membrane, the transparent film surrounding the cube PBNPs can be observed which shows that the hybrid membrane successfully wraps the PC inside, and the particle size of PCM is stable at about 100nm (Fig. 1b). In addition, through DLS analysis, it was detected that the average hydration diameters of PB, PC and PCM are 98.49 nm, 110.4 nm and 128.3 nm respectively (Fig. 1c). The zeta potential measurement showed the surface of PB is loaded with negative charge. Due to the positive charge on the surface of CS-5, after loading with CS-5, potential of PC increased significantly from -25.4 ± 1.14 mV to -10.1 ± 0.08 mV (Fig. 1d); UV-vis spectra indicates that PC had two absorption peaks, one is the absorption peak of PB at 700 nm, and the other is the absorption peak of CS-5 at 372 nm (Fig. 1e). This result shows that CS-5 is successfully loaded into PB. The UV-vis spectrum of PCM displays the characteristic peak of RBC-HCTm at 416 nm (Fig. 1d). Moreover, coating with RBC-HCTm directly results in the potential reduction of PCM (Fig. 1d, e).

Monitoring the stability of PCM in 10% FBS and PBS for 6 consecutive days, we found that the average particle size of PCM is stable between 120nm and 130nm (Fig. 1f), indicating that PCM has an excellent stability. When the mass ratio of PB to CS-5 was 1:1, the encapsulation efficiency and loading efficiency of PB to CS-5 are approximately 60 %, which indicates that CS-5 has a high drug loading efficiency of in PB (Fig. S1a). We further investigated the effects of laser and pH value on the release of CS-5 from PCM. The results indicates that the cumulative release of CS-5 in PCM with pH 7.4 was only 46.19% after 72 hours, while the cumulative release of CS-5 reached 97.59% after pH 5.4 plus laser irradiation (Fig. S1b). At the same time, the laser can significantly stimulate the release of CS-5 is also verified in Fig. S1c. After RBCm and HCTm are labeled with DiI and DiO respectively, and the fusion of RBCm and HCTm is observed under laser scanning confocal microscope, the yellow fluorescence indicates the successful fusion of RBCm and HCTm (Fig. 1g). In addition, SDS-PAGE results shows that the integrity of membrane protein in PCM was consistent with that of other groups (Fig. 1h).

3.2. Photothermal performance characterization

Photothermal performance of PB is crucial for tumor PTT[36]. The temperature rising curves of PBS, PB, PC, and PCM under laser irradiation (808 nm, 1.2 W/cm²) for 5 min were compared. Except PBS, the temperature , of PB, PC, and PCM increases from 27℃ to 49.5 ℃, higher than the temperature required to kill tumor cells (42 ℃) [37] (Fig. 2a, b). As the same time, CS-5 and hybrid membrane had no effect on the photothermal properties of PB. However, the photothermal effect of PCM is obviously dependent on its concentration (Fig. 2c). Furthermore, the photothermal conversion efficiency of the PCM is 31.33% (Fig. 2d), which is higher than SQ1 nanoprobe (η = 25.6%)[38] and Au nanorods (η = 23.7%), respectively [39]. The long-term photothermal effect of PB on tumor sites has great potential for its application in targeted tumor therapy [40]. Photothermal stability assay indicated that the maximum temperature peak and the UV-vis spectrum of PCM remain unchanged after six cycles of laser on/off irradiation (Fig. 2e, f). In addition, the release rate of CS-5 under 808 nm laser irradiation was increased significantly (44.1% vs 7.56%) (Fig. S1c). The above results demonstrated that PB is an excellent photothermal agent for PTT due to its excellent photothermal properties and stability. It suggests that it has a good prophase of application in tumor treatment.

3.3. Biocompatibility of the PCM in vitro

Although FDA has proven that PB has an excellent safety as an antidote[41], biomembrane with excellent biocompatibility also contribute to the clinical transformation of nanomaterials[42, 43]. However, it is still necessary to analyze the effects of PCM on cytotoxicity, hemolysis, and coagulation in vitro, so as to provide pre-validation for the application in vivo. The cytotoxicity analysis of PB and PBM on HCT116 cells, SW480 cells, VSMC cells, and NIH-3T3 cells indicates that even if the concentration of PB was as high as 100 µg/mL, the cell survival rate of PBM group was as high as to 85%, which significantly higher than that of PB group (80%)(Fig. 3a, b). In addition, compared with the positive group, the other groups have no effect on hemolysis rate, in which PCM is of 2.97 %, and PC is 4.12 %, even at the highest concentration of PB with 200 μg/mL, there is no significant changes in erythrocyte morphology (Fig. 3c, d). There is also no difference in coagulation rate between different materials groups and the PBS groups (Fig. 3e), these results also indicate that different materials have no significant effect on hemolysis and coagulation, and also show that PCM has an excellent biocompatibility, and has no toxic side effects, which is conducive to further clinical trials in vivo.

3.4. Cell uptake and immune escape assay

Cell uptake efficiency and immune escape assay were used to evaluate the practical potential of PCM in vivo. As shown in Fig.S2a, red fluorescence intensity in the cytoplasm represents the uptake of substances by cells. The results shows that the uptake of PBM in macrophages was significantly lower than that of PB (p < 0.001 and p < 0.01) when PBM and PB at the same concentration of 50 µg/mL and 100 µg/mL. As shown in Fig. S2b, the low uptake of PCM in macrophages revealed that the weak immune response is caused by the immune escaping ability of the hybrid membrane, thus prolonging its time in the blood circulation. In addition, by evaluating the uptake of PBM in HCT116 cells at different time point, it was found that the red fluorescence gradually increased with time, but remained unchanged after 6 hours (Fig. S2c). Therefore, the optimal laser irradiation time was determined at 6 hours to ensure the maximum photothermal efficiency.

3.5. Anti-tumor effect and mechanism in vitro

First of all, we investigated the photothermal killing effect of PB on HCT116 cells when the concentration of PB was 30 μg/mL, 40 μg/mL and 50 μg/mL, which is shown in Figure.S3. The kill effects of PB at different concentrations are 16.6%, 59.6% and 83.3%, respectively, indicating that 30μg/mL of PB was the optimal concentration for photothermal-assistant chemotherapy. Therefore, the selected concentration is 30μg/mL of PB for subsequent experiments. Then, the cell killing rates of PB + L, PC, and PC + L (PB=30μg/mL, PB: CS-5=15:1) on HCT116 cells were measured respectively. The results indicated that the combined efficacy of PC and laser (85.3 %) was better than that of stacking effect of PC (63.9 %) or PB plus the laser (16.6 %) (Fig.4a). In additional, compared with PC + L, the cell killing rates of PCM + L is slightly reduced, which may help the slow release of CS-5 from PC after coating with hybrid membrane (Fig.4b). In addition, the effect of chemo-photothermal therapy of PCM in vitro was further verified by living/dead staining, in which green fluorescence and red fluorescence represented living cells and dead cells respectively. Compared with other groups, strong red fluorescence reflected the significant killing effect of PC or PCM plus laser on tumor cells, which also confirmed with the MTT results (Fig.4c). The main anti-cancer mechanism of CS-5 is to induce tumor cell apoptosis[11, 44]. P53 can mediates cell apoptosis by regulating Bcl-2 proteins such as Bcl-2 and Bax[45].The low expression of the proapoptotic protein of Bcl-2 releases their restricted anti-apoptotic protein of Bax to initiate apoptosis, and Bax induces the release of apoptotic factors such as Caspase-3 to mediate the final cells death [46]. By investigating the expression levels of apoptosis-relate proteins in HCT116 cells, we found that CS-5 significantly induced the expression of pro-apoptotic protein C-Caspase-3, P53, and Bax, and down-regulated the expression level of anti-apoptotic protein Bcl-2 (Fig.4d, e). These results suggested that CS-5 played its role in colon cancer mainly by inducing the apoptosis of HCT116 cancer cells.

3.6. Ferroptosis mechanism in vitro

Recent studies have shown that CS-5 can induce ferroptosis[12], which is the characterized by including ROS through Fenton reaction and inhibiting the expression of MMP and GPX4 protein[17, 47]. Therefore, we used JC-1 assay to detected the MMP protein expression. Red fluorescence and the green fluorescence represented the normal MMP and the decreased MMP, respectively. The decrease of red fluorescence and increase of green fluorescence enhancement in PCM + L group proved that CS-5 and PB plus laser can promote cell ferroptosis by inducing mitochondrial damage (Fig. 5a). Meanwhile, fluorescence imaging indicated that CS-5 and PB plus laser could induce ROS generation. In addition, PCM showed the most significant tumor cell killing effect under laser induction (Fig. 5b).

Research have reported that inhibiting the expression of HSP70 protein can improve the sensitivity of cancer cells to PTT, thus improving the efficacy of tumor cell ablation[48]. Therefore, we detected the effect of PB on the expression of HSP70 protein, and found that laser irradiation of PB with can significantly reduce the expression level of HSP70 protein in HCT116 cells (Fig. 5c), which can greatly enhance the efficacy of PTT in the treatment of colorectal cancer. In addition, HIF-1a can reduce iron and ROS levels, which implies that HIF-1a has a universal role in the anti-ferroptosis of other cancers[49, 50]. PCM with laser can effectively reduce the expression of ferroptosis-related proteins of GPX4 and HIF-1a protein (Fig. 5d). All the results indicated that PCM plus laser had the remarkable effect on ferroptosis by down-regulation the expression of GPX4 and HIF-1a protein, inducing ROS production, and reducing MMP.

3.7. Biodistribution and pharmacokinetics in vivo

In order to comprehensively evaluate the homotypic targeting ability of the hybrid membrane and metabolism of PB in vivo, 100µL of PB^Ce6 and PB@M^Ce6 ( 5 mg/kg PB was used) were injected into tumor-bearing mice via tail vein, and the fluorescence signal distribution of Ce6 in tumor-bearing mice was monitored at different time points. As shown in Fig 6a, the fluorescence signal of Ce6 was note found at the tumor site of mice administered PB^Ce6 after 4 h, but remained unchanged after 48 h in PB@M^Ce6group. After 48 hours, the tumors and main organs were collected from mice for fluorescence imaging. As shown in Figure 6b, the fluorescence signals are enriched in the tumor site of the mice, and the fluorescence intensity of PB@M^Ce6 was significantly stronger than that of PB^Ce6 (P < 0.05). This result demonstrated the excellent tumor-targeting ability of HCTm and long half-life time of RBCm. In addition, we found that the strong fluorescence signal of PB^Ce6 and PB@M^Ce6 in liver and kidney, as they were mainly metabolized through liver and kidney (Fig. 6c-d). Fig. 6e-f show that the average serum half-life of Ce6, PB^Ce6 and PB@M^Ce6 was 0.64 h, 1.43 h and 1.63 h, respectively, indicating that RBCm could assist nano-drugs escape the clearance of macrophages and prolong the half-life in vivo.

3.8. Anti-tumor effect in vivo

The chemotherapy/hyperthermia scheme of PCM on the mouse model of colorectal cancer is shown in Fig. 7a. When the tumor volume grew to 100 mm³, all mice were randomly divided into five groups: (1) PBS, (2) CS-5, (3) PBM + L, (4) PCM, (5) PCM + L. First, we evaluated the photothermal conversion performance of different nanomaterials in vivo. After the tumor was irradiated by laser (808 nm, 1.2w/cm²) for 5 min, the temperature of tumor region with PBM and PCM administration reached to 49.5℃ (Fig. 7b), indicating the excellent photothermal conversion performance and tumor targeting of PCM in vivo. On the contrary, mice treated with PBS did not find temperature changes. After 8 cycles of treatment, the tumor size of PBS group gradually increased, and the maximum volume of tumor was about 1.800 cm³. Compared with PBS group, the tumor growth of CS-5, PBM + L, and PCM groups was slightly inhibited, with the inhibition rates of 41.16%, 31.27%, and 66.04%, respectively. The tumor growth in PCM + L group was effectively controlled (P < 0.0001) with the highest inhibition rate of 87.97% (Fig. 7c). The change trend relative tumor volume and tumor weight in vivo (Fig. 7d-f and Fig. S3) is consistent with the therapeutic effect, and the body weight of mice hao no significant changes (Fig. 7g). These results proved the combine effects of PCM with laser on tumor growth inhibition.

To intuitively observe the anti-tumor mechanism in vivo of PCML intuitively, we performed HE, TUNEL, and immunohistochemical staining on tumor sections. HE staining showed that a large number of necrotic tumor regions were observed in PCM + L group. In TUNEL staining, comparing with other groups, PCM + L group showed the strongest green fluorescence, indicating apoptotic cells, (Fig. 7h). At the same time compared with PBS group, the protein expression levels of Ki67, GPX4, and SLC7A11 in tumor tissues of PCM + L group were significantly decreased. This result also confirmed that laser PCM can effectively inhibit tumor growth by inhibiting the expression of Ki67 protein. In addition, the experimental results in Figure 7i indicated that CS-5 could downregulate the expression of GPX4 and SLC7A11 protein to induce ferroptosis in HCT116 cells, which also contribute to the tumor growth inhibition. To sum up, PCM can achieve satisfactory effect in chemo-photothermal therapy of colorectal cancer by inducing ferroptosis and apoptosis of HCT116 cells.

3.9. The comprehensive landscape of gut microbiota

The changes in intestinal microbiota may be related to the curative effect of colon cancer therapy[51, 52]. Therefore, 16S ribosomal RNA (rRNA) sequencing analysis was performed to investigate the change of intestinal microbiota after PCM plus Laser-induced treatment. Figure 9a indicated that the control group, PBS and PCML group share 353 of the total 2727 OTUs, PBS and PCML group share 885 OTUs, and the 485 OTUs aue unique to the PCML group. The alpha diversity of intestinal microbiota was evaluated using Chao1 and OTU indexes, and the results showed that species diversity the PBS and PCML groups were significantly higher than that of the Control group (Fig. 9b-c). The abundance distribution of intestinal microbiota was demonstrated with circos plot and the heatmap(Fig. 9d-e). Compared with the control group and PBS group, the abundance of Helicobacte pylori, Ricardia, and Streptococcus in the PCM+L group increased, while the abundance of Candidatus saccharimonas, anaerotruncus, HT002, and Enterorhabdus decreased. Moreover, the indicator analysis was conducted to screen indicative microbiota biomarkers. Compare with the control group, Chloroflexi, Myxococcota, Gemmatimonadota, WPS-2, Campylobacterota, and Proteobacteria were significantly enriched in PBS and PCML groups, which may serve as biomarkers for colon cancer detection. Nitrospirota and NB1-j were significantly enriched in PCML group, which may serve as a biomarker for evaluating the efficacy of tumor therapy (Fig. S4a), and a branch map is drawn to identify specific intestinal microflora (Figure S4b). In addition, the PICRUSt2 method was used to predict the microbial population function in the PCML treatment group across the pathway database (Fig. S4c). The results indicated that pentose phosphate pathway and superpathway of pyrimidine deoxyribonucleotides de novo biosynthesis are significantly related to the PCML with the potential of anticancer drug target.

3.10. Biosafety assessment in vivo

In order to highlight the clinical application of PCM, HE staining, blood routine and biochemical analysis were used to evaluate the biological safety of PCM in major organs (heart, liver, spleen, lung, and kidney). HE staining showed that different drug samples had no- obvious toxic and side effects on main organs except that the cardiac toxicity of CS-5 might slightly increase the myocardial clearance compared with PBS group (Fig. 8a). There were no differences in routine blood and biochemical tests between different groups, except that red blood cells and platelets could slightly increase in CS-5 group compared with PBS group, which may relate to the poor prognosis of drug Chemotherapy (Fig. 8b-f). In short, the PCM for chemo/thermotherapy has high biological safety and has the potential to be developed into clinical nanomedicine.

In this study, we constructed a biomimetic nanomedicine (PB@CS-5@M), which has good biocompatibility, tumor-targeting and laser-responsive properties, and can be used effectively for chemo/thermotherapy against colon cancer. The nanomedicine has the following prominent advantages: (1) The biomimetic hybrid membrane of RBCm-HCTm could prolong the blood circulation time of the nanomedicine, target the tumor site accurately, and reduce the cardiac toxicity of CS-5 in chemotherapy. (2) The nanomedicine can be used as ferroptosis inducers to down-regulate the expression of GPX4 and HIF-1a protein, up-regulate ROS production, and reduce mitochondrial membrane potential. (3) The high temperature caused by laser stimulation can trigger PB to rapidly release CS-5 and iron and ferrous ions, which further improves the therapeutic effect. To sum up, the chemotherapeutic effect of CS-5 combined with the photothermal effect of PB can effectively treat colon cancer by inducing ferroptosis. However, the precise dosage of CS-5 needs further optimization.

Author Contributions Statement

Y.W. and J.T were responsible for experimental design, overall experimental verification, data process and article writing. M.L. and Z.W. revised and verified the experimental data. R.C. and C.F. drawn the schematic diagram and were responsible for the beautification of all figure. J.Y. and Q.P. were responsible for drug efficacy verification in vitro. R.L. and B.L. was responsible for experimental design, format modification and manuscript revision.

Ethics approval and consent to participate

All animal experiments were carried out at the Laboratory Animal Center of Hunan Normal University School of Medicine, and all experiment protocols conform to the principle “Declaration of Helsinki of the Ministry of Health” (approval No. D2022012). All authors volunteered to participate in the study, and all animal ethics experiments were conducted with their consent and they could be withdrawn at any time. All authors will receive a copy of this consent.

Consent for publication

All authors agree to the publication of this article.

Availability of data and materials

All data and materials are original and true.

Competing interests

The authors show that there is no conflict of interest

Funding

We thank the support of grants from the Hunan Provincial Key Research and Development Program (No. 2019SK2042), the National Natural Science Foundation of China (No. 31072141, No.12034006) and the Applied Basic Research Project of Changzhou Science and Technology Bureau (No. 202202086).

Authors' contributions

The author participated in the design, experiment and writing of the manuscript.

Acknowledgements

We sincerely thank all funding providers, research fund projects, and the experimental platform provided by the College of Biology, Hunan University.

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Bufotalin-loaded biomimetic prussian blue nanoparticles for colorectal cancer chemo-photothermal therapy via induce ferroptosis

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