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Tumor microenvironment responsive Mn3O4 nanoplatform for in vivo real-time monitoring of drug resistance and photothermal/chemodynamic synergistic therapy of gastric cancer | Journal of Nanobiotechnology

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Accurate monitoring of MDR is very important for individualized treatment of gastric cancer. There have been methods to assess MDR in past studies, such as fresh tumor cell culture tests, cancer biomarker tests, or in vivo tumor imaging to assess the degree of MDR. However, to a certain extent, these methods cannot reflect the real state of MDR in real time. Therefore, it is necessary to explore new methods for real-time monitoring of MDR. Once MDR is monitored, individualized treatment can be performed on MDR gastric cancer patients to improve their survival rate. In view of this, a GMBP1 cross-linked self-enhanced nanoplatform of Mn3O4 NPs with a GSH consumption function was established, which can react with GSH to generate Mn2+ to enhance T1-weighted MRI, thereby mediating in vivo MDR monitoring. As a Fenton-like reagent, Mn2+ can convert H2O2 in cells into highly toxic ·OH, which can exert a CDT effect. Mn3O4 NPs can also be used as photothermal conversion agents for PTT, which can effectively improve Fenton reaction efficiency and the production of ·OH, thereby enhancing the CDT effect. MPG NPs achieved in vivo CDT/PTT synergistic therapy, providing a good nanoplatform for the treatment of MDR gastric cancer (Fig. 1).

Fig. 1
figure 1

The mechanism of pH/H2O2/GSH-responsive MPG NPs as a multifunctional self-enhanced nanoplatform for gastric cancer MDR monitoring and CDT/PTT synergistic therapy. After endocytosis, MPG NPs can react with intracellular GSH by redox reaction to produce Mn2+, which has Fenton-like activity and can convert endogenous H2O2 into highly toxic ·OH under HCO3 conditions. Mn2+ can enhance MRI for in vivo MDR monitoring. Under laser irradiation, MPG NPs can perform CDT/PTT synergistic therapy of MDR in gastric cancer

Preparation and characterization of MPG NPs

The synthesis scheme of MPG NPs is shown in Fig. 2A. Among them, Mn3O4 NPs were synthesized according to the thermal decomposition method described in a previous study [30]. As shown in the TEM image of Fig. 2B, the Mn3O4 NPs dispersed in the organic solvent present a spherical structure with uniform particle size, good dispersibility, and a particle size of approximately 7.4 ± 0.21 nm. The Mn3O4 NPs can be well dispersed in water after being coated with PDA. The particle size of Mn3O4 NPs coated with the PDA layer is slightly larger, approximately 8.2 ± 0.27 nm, and the PDA coating on the surface can be observed clearly (illustration) (Fig. 2C). The crystal form of Mn3O4 NPs was verified by XRD (Fig. 2D). The results show that the diffraction peaks of the synthesized Mn3O4 NPs are basically coincident with the diffraction peaks of Joint Committee on Powder Diffraction Standards (JCPDS) Card No. 24–0734, which belong to the tetragonal system. The synthetic method of tetrazole compound T is shown in Additional file 1: Fig. S1A. To verify the successful synthesis of T, the mass spectrum (Additional file 1: Fig. S1B) and 13 C NMR spectrum (Additional file 1: Fig. S1C) of T were obtained. ESI-MS: m/z 339.1 [M + H]+; 13 C NMR (150 MHz, DMSO-d6) δ = 38.45, 38.59, 55.06, 114.47, 121.08, 125.75, 135.89, 159.83, 163.05, 164.19, 164.98. The amino group of T and the oxidation state quinone on the surface of PDA form a covalent graft through the Schiff base reaction. The reaction easily proceeds without complicated equipment and harsh conditions [31]. As shown in the ultraviolet absorption spectrum of Fig. 2E, tetrazole compound T has a maximum absorption peak at 290 nm. After the photoclick reaction between T and GMBP1-Ack, there is a maximum absorption peak at approximately 370 nm. This is due to the formation of new pyrazoline products after the photoclick reaction [32]. Mn3O4@PDA NPs have no characteristic peak of ultraviolet absorption. Mn3O4@PDA-T NPs had the maximum absorption peak at approximately 285 nm, which proved the successful crosslinking of T. After the photoclick reaction between Mn3O4@PDA-T and GMBP1-Ack, there was a maximum absorption peak at approximately 365 nm. The blueshift of the absorption of Mn3O4@PDA-T NPs may be caused by the steric hindrance of PDA. These results initially proved the successful synthesis of MPG NPs. As shown in the fluorescence spectrum of Fig. 2F, under 410 nm laser excitation, the maximum emission wavelength after the photoclick reaction of T and GMBP1-Ack is approximately 535 nm, and the maximum emission wavelength of MPG NPs is approximately 545 nm. This is due to the fluorescent pyrazoline product produced by the photoclick reaction. As shown in Fig. 2, G and H, the hydrated particle size and zeta potential of Mn3O4@PDA NPs, Mn3O4@PDA-T NPs and MPG NPs were measured by a Malvern particle size analyzer. The results show that the hydrated particle size of Mn3O4@PDA NPs is approximately 32.7 ± 4.8 nm, and the potential is −15 mV. The negative potential of Mn3O4@PDA NPs is attributed to the quinoneimine and catechol groups on the surface of PDA. The reversible dissociation and deprotonation/protonation of the amine and catechol groups make PDA generate a positive or negative charge, and the net charge is negative [33]. After crosslinking T on the surface of Mn3O4@PDA NPs, the hydrated particle size increases to approximately 37.8 ± 5.6 nm, and the potential tends to be neutral. After the photoclick reaction between Mn3O4@PDA-T NPs and GMBP1-Ack, the particle size continues to increase to approximately 50.5 ± 3.7 nm, which is due to the influence of the surface hydration layer, and the potential has also become + 3 mV. The stability of nanoparticles is an important factor for in vivo long-term imaging studies. The MPG NPs were dispersed in PBS and 10% FBS, and the changes in particle size at different temperatures were measured to verify the stability of the MPG NPs. As shown in Fig. 2I, the particle size of MPG NPs did not change significantly within 6 days, and the solution remained clear. The results indicate that MPG NPs have good stability.

Fig. 2
figure 2

Characterization of MPG NPs. A Synthesis of MPG NPs. B TEM of Mn3O4 NPs. C TEM of Mn3O4@PDA NPs. D XRD spectrum of Mn3O4 NPs. E UV absorption spectra of Mn3O4@PDA NPs, T, Mn3O4@PDA-T NPs, T-GMBP1, and MPG NPs. F Fluorescence spectrum of T-GMBP1 and MPG NPs. G The size of Mn3O4@PDA NPs, Mn3O4@PDA-T NPs and MPG NPs. H The zeta potential of Mn3O4@PDA NPs, Mn3O4@PDA-T NPs and MPG NPs. I The size change of MPG NPs in different solvents at different temperatures

In vitro MRI and fluorescence imaging of MPG NPs

To test the MRI contrast ability of MPG NPs, the relaxation characteristics of Mn3O4@PDA NPs and MPG NPs were measured with MRI scanner (0.5 T). As shown in Fig. 3A, as the concentration of Mn3O4 NPs increased, Mn3O4@PDA NPs and MPG NPs both showed signal enhancement in the T1-weighted MRI image. According to the linear fit of 1/T1 to the concentration of Mn3O4 NPs, the r1 values of Mn3O4@PDA NPs and MPG NPs were calculated to be 0.606 mM− 1s− 1 and 0.461 mM− 1s− 1, respectively (Fig. 3B). PDA modification enhances the T1 relaxivity of Mn3O4 NPs, and its r1 value is more than 3 times higher than that of Mn3O4@PEG NPs (0.2 mM− 1s− 1) [7], confirming the potential use of Mn3O4@PDA NPs as MRI imaging agents. The r1 value of MPG NPs is lower than that of Mn3O4@PDA NPs. The main reason for this result is that the connection of T on the surface of PDA reduces the contact of water protons with Mn3O4 nuclei, thereby weakening the T1 relaxation properties of NPs. Although the r1 value of MPG NPs is reduced, it is still higher than that of Mn3O4@PEG NPs, indicating that it has the ability for in vivo MRI. At the same time, the in vitro fluorescence imaging performance of MPG NPs was tested by the PerkinElmer IVIS system. As shown in Fig. 3A, as the concentration of MPG NPs increased, the fluorescence signal increased, which was consistent with the result of fluorescence signal extraction in the region of interest (Fig. 3B), indicating that GMBP1 successfully crosslinked with Mn3O4@PDA NPs through the photoclick reaction.

Fig. 3
figure 3

In vitro MRI and fluorescence imaging of MPG NPs. A MRI and fluorescence imaging of different concentrations of Mn3O4@PDA NPs and MPG NPs. B MRI relaxation rate and fluorescence intensity of Mn3O4@PDA NPs and MPG NPs at different concentrations. C MRI of different concentrations of MPG NPs at different pH values. D MRI relaxation rate of MPG NPs at different concentrations and different pH values. E MRI of different concentrations of MPG NPs with H2O2 at different pH values. F MRI relaxation rate of MPG NPs with H2O2 at different concentrations and different pH values. G MRI of different concentrations of MPG NPs with or without GSH. H MRI relaxation rate of MPG NPs with or without GSH at different concentrations

Previous studies have shown that pH or GSH can enhance T1-weighted MRI of Mn3O4 NPs [27, 34, 35] because Mn3O4 can generate Mn2+ in response to the TME. To verify whether MPG NPs can respond to the TME, the pH sensitivity of MPG NPs was first verified. During tumor growth, glycolysis and metabolism are upregulated to produce a large amount of lactic acid, resulting in a lower pH in tumor tissues and weak acidity of the TME [36]. To simulate the blood environment and TME, the pH values were set to 7.4, 6.0 and 5.0 [37]. The relaxation images and relaxation times of MPG NPs at different pH values were measured, and the relaxation rate was calculated. As shown in Fig. 3C, under the same pH conditions, with increasing Mn3O4 concentration in MPG NPs, the MRI signal gradually increased. At the same Mn3O4 concentration, the lower the pH value, the stronger the MRI signal. Figure 3D shows the relaxation rate at different pH values. The results show that the relaxation rate r1 is only 0.505 mM− 1s− 1 at pH 7.4 and 0.779 mM− 1s− 1 at pH 6.0. When the pH value is 5.0, the relaxation rate can reach 1.321 mM− 1s− 1, which is 2.6 times that under neutral conditions. These results indicated that the synthesized MPG NPs are sufficiently sensitive to acidic environments. Under acidic conditions, the MRI signal increases significantly, the relaxation time becomes shorter, and the relaxation rate increases. Furthermore, MPG NPs can be used as excellent pH-responsive T1-weighted MRI agents.

Furthermore, H2O2 is overproduced in malignant tumor cells, leading to a significant increase in H2O2 in the TME [38]. There have been reports of H2O2 dissociating MnO2 into O2 and Mn2+ under weak acid conditions to enhance T1-weighted MRI [39, 40], but there is no report in terms of Mn3O4-related research. Therefore, the T1-MRI performance of Mn3O4 under H2O2 conditions was explored. In Fig. 3E, it is observed that MPG NPs have strong concentration-dependent signal enhancement. In addition, the relaxation rate of MPG NPs at pH 6.8 (r1 = 1.009 mM− 1s− 1) is 1.9 times that at pH 7.4 (r1 = 0.5063 mM− 1s− 1) (Fig. 3F), indicating that protons are essential in the decomposition of Mn3O4 induced by H2O2. At the same time, after adding H2O2, the T1 enhancement obtained in the presence of H+ is also attributed to the catalytic reaction of H2O2 reduction [41, 42].

In addition, GSH in tumor tissue is 5 times that of normal tissue, and GSH plays a key role in protecting cells from various harmful substances (such as H2O2, superoxide, ·OH and other active substances) [12]. To evaluate the effectiveness of MPG NPs as GSH-activated T1 contrast agents, MPG NPs were dispersed in a solution containing GSH (15 mM) and incubated for 5 min, and T1-weighted MRI images were obtained (Fig. 3G). In the presence of GSH, T1-weighted MRI images of MPG NPs showed stronger signal enhancement than MPG NP solutions without GSH, and the r1 relaxation rate was increased by 7.4 times (Fig. 3H). The enhancement of the relaxation rate is due to the release of Mn2+ after the redox reaction between Mn3O4 NPs and GSH, and the water coordination number in the center of Mn increases, which leads to the enhancement of the T1-weighted MRI signal [27]. The above results proved that MPG NPs have the potential to respond to TME and enhance MRI, which can be used as good self-enhanced nanoplatforms.

In vitro chemodynamic activity of MPG NPs

The occurrence of MDR is closely related to TME. To verify whether MPG NPs can respond to the TME and exert in vivo CDT effect, the chemodynamic activity of MPG NPs was studied in vitro. First, it was verified that Mn2+ can react with H2O2 to produce ·OH through a Fenton-like reaction in the presence of HCO3, thereby degrading methylene blue (MB). It can be seen from Fig. 4A and B that when Mn2+ and H2O2 are incubated in NaHCO3/CO2 buffer for 30 min, the absorbance of MB at 665 nm is significantly reduced, and the presence of Mn2+ or H2O2 alone has no significant effect on the absorbance of MB. In aqueous solution, the absorbance value of MB did not change significantly. As the concentration of H2O2 increased, the absorbance of MB gradually decreased (Fig. 4C). These results show that the Mn2+-mediated Fenton-like reaction can effectively produce ·OH under physiological NaHCO3/CO2 conditions, which is consistent with previous research [12]. In Fig. 4D, in the presence of 10 mM GSH, the absorbance value of MB did not significantly decrease, which indicates that ·OH-induced MB degradation was significantly inhibited in the presence of 10 mM GSH. With increasing GSH concentration (0–10 mM), the absorbance value of MB gradually increased, indicating that the degradation of MB was gradually inhibited (Fig. 4E). The remaining amount of MB in each group is shown in Additional file 1: Fig. S2A. Next, the chemodynamic activity of Mn3O4@PEG NPs was verified. In previous studies, the color of the MnO2 solution gradually disappeared after gradually adding different concentrations of GSH solution, and it was proven that MnO2 reacted with GSH to produce Mn2+ and GSSG. Different concentrations of GSH solution were gradually added to the Mn3O4@PEG NP solution, and the color of the solution gradually became lighter (Additional file 1: Fig. S2B), and the reaction efficiency with GSH was significantly higher than that of pH-triggered reactions (Additional file 1: Fig. S2C), which is consistent with a previous study [12]. Figure 4F shows that as the concentration of GSH in the Mn3O4@PEG NP solution increases, the absorbance value of MB gradually decreases. When the concentration of GSH reaches 12 mM, the absorbance value of MB gradually increases, indicating that Mn3O4 NPs react with GSH to produce Mn2+, which reacts with H2O2 to produce ·OH through a Fenton-like reaction. However, with the increase in free GSH, the ·OH that produced by the Fenton-like reaction is gradually eliminated, thereby inhibiting the degradation of MB (Fig. 4G). Figure 4H shows the degradation of MB after adding different concentrations of GSH to the MPG NP solution. The results show that as the concentration of GSH increases, the absorbance of MB decreases first and then increases gradually, which is consistent with the above results [12]. The degradation efficiency of MB by ·OH in the presence of GSH is shown in Additional file 1: Fig. S2, D to F. The results show that when the GSH concentration was as high as 10 mM, Mn3O4@PEG NPs still showed 57.69% MB degradation efficiency, which is approximately 3 times the MB degradation efficiency promoted by Mn2+ (18.8%) under the same conditions. MPG NPs showed 39.92% MB degradation efficiency, which is approximately 2.12 times the MB degradation efficiency promoted by Mn2+ under the same conditions. These results indicate that MPG NPs have good in vitro chemodynamic activity and have the potential for in vivo CDT.

Fig. 4
figure 4

In vitro chemodynamic activity of MPG NPs. A UV-visible absorption spectra and photo (inset) of MB degradation induced by Mn2+ in different solutions. B UV-visible absorption spectra and photo (inset) of MB in the presence of Mn2+ and H2O2 alone. C UV-visible absorption spectra and photo (inset) of MB degradation induced by Mn2+ when the concentration of H2O2 gradually increases. D UV-visible absorption spectra and photo (inset) of MB degradation induced by Mn2+ with or without GSH. E UV-visible absorption spectra and photo (inset) of MB degradation induced by Mn2+ when the concentration of GSH gradually increases. F UV-visible absorption spectra and photo (inset) of MB degradation induced by Mn2+ (Mn3O4@PEG) when the concentration of GSH gradually increases. G UV-visible absorption spectra and photo (inset) of MB degradation induced by Mn2+ (Mn3O4@PEG) when the concentration of GSH gradually increases. H UV-visible absorption spectra and photo (inset) of MB degradation induced by Mn2+ (MPG NPs) when the concentration of GSH gradually increases. I Schematic diagram of MPG NPs reacting with GSH to produce Mn2+ and converting endogenous H2O2 into ·OH. J DCF fluorescence of SGC 7901 ADR with or without MPG NPs

The above experiments have proven that Mn3O4 NPs can generate Mn2+ in the presence of GSH and enhance MRI. Mn2+ can convert H2O2 into highly toxic ·OH to kill tumor cells (Fig. 4I). The production of ROS in the presence of MPG NPs was verified in the cell. Figure 4J shows the observation of ROS production by detecting cell DCF fluorescence after adding different materials. DCFH-DA has no fluorescence and is hydrolyzed into DCFH after entering cells and then rapidly oxidized to DCF with fluorescence [43]. The fluorescence intensity of DCF shows that in the presence of manganese ions, the fluorescence of DCF is significantly stronger than that of the control group. The quantitative analysis of the mean fluorescence intensity (MFI) is shown in Additional file 1: Fig. S2G. The results showed that the DCF fluorescence intensity of cells coincubated with MPG NPs was significantly different from that of the other groups. This indicates that MPG NPs react with GSH in cells to generate Mn2+, which mediates the Fenton-like reaction and generates ROS. It not only reduces the elimination of ·OH by GSH but also produces more ROS and enhances the killing effect on tumor cells.

In vitro and in vivo photothermal effect of MPG NPs

Studies have shown that Mn3O4 NPs have high molar extinction coefficient and strong absorption in the near-infrared region [28, 44], which can be used for in vivo PTT. In addition, PTT can increase the efficiency of the Fenton reaction and the productivity of ·OH by locally heating the tumor [13]. To verify whether MPG NPs can be used for in vivo PTT, the photothermal effect of MPG NPs was verified. First, it was determined whether Mn3O4 NPs have photothermal effects and enhanced in vivo PTT. The temperature changes of different concentrations of Mn3O4@PEG NPs irradiated with 808 nm lasers with different powers for 5 min were measured. As shown in Fig. 5A, under laser irradiation (1 W), the temperature of the aqueous solution does not change much. As the Mn3O4@PEG NP concentration increases, the temperature gradually increases. As shown in Fig. 5B, as the laser power increases, the temperature of the Mn3O4@PEG NP solution (1 mg mL− 1) gradually increases. These results indicate that Mn3O4@PEG NPs have certain photothermal properties. As shown in Fig. 5C, the temperature change of the solution of the same concentration of Mn3O4@PEG NPs, PDA and MPG NPs (1 mg mL− 1) was measured after 808 nm laser irradiation (2 W) for 5 min. The results show that the temperature of MPG NPs increases the fastest, and the temperature can reach 55 °C after 5 min. Compared with Mn3O4@PEG NPs and PDA, MPG NPs have better photothermal performance. To detect the influence of laser power on the photothermal effect of MPG NPs, the temperature change of MPG NPs under different laser powers for 5 min was measured. As shown in Fig. 5D, when the laser power is 0.5 W, the temperature of the MPG NP solution does not change much. As the power increases, the temperature gradually increases. When the power reaches 2 W, the temperature of the MPG NP solution can reach 55 °C after 5 min of irradiation. To verify the influence of the concentration on the photothermal effect of MPG NPs, the temperature changes of MPG NP solutions of different concentrations under 808 nm laser irradiation (1 W) for 5 min were measured. The solution without MPG NPs was used as a control. As shown in Fig. 5E, after 5 min of laser irradiation, the temperature of the control solution did not change much, and the temperature gradually increased as the concentration increased. To verify whether MPG NPs have good photothermal cycling properties, the MPG NP solution was first irradiated with a laser (2 W) for 5 min, and then irradiation was stopped and returned to room temperature. The cycle was repeated 5 times. As shown in Fig. 5F, the temperature of the MPG NP solution can rise above 50 °C after each irradiation and return to room temperature within a certain period of time. The results show that laser irradiation does not affect the photothermal performance of MPG NPs.

Fig. 5
figure 5

In vitro and in vivo photothermal effect of MPG NPs. A Temperature changes of different concentrations of Mn3O4@PEG after 5 min of laser irradiation. B Temperature change of Mn3O4@PEG after 5 min of laser irradiation with different powers. C Temperature change of the same concentration of Mn3O4@PEG, PDA and MPG NPs after 5 min of laser irradiation. D Temperature change of MPG NPs after 5 min of laser irradiation with different powers. E Temperature change of different concentrations of MPG NPs after 5 min of laser irradiation. F Temperature change of MPG with or without laser irradiation. G Temperature changes under laser irradiation after 6 h of in vivo injection of MPG NPs. H Schematic diagram of the photothermal effect of MPG NPs under laser irradiation after endocytosis

To verify the in vivo photothermal effect of MPG NPs, photothermal images of the tumor-bearing mice before and after laser irradiation were obtained. Figure 5G shows that the temperature of the tumor site of the mice in the PBS injection group did not increase significantly after laser irradiation and only increased from 35.4 to 37.9 °C. The tumor site temperature of mice injected with Mn3O4@PDA NPs increased from 35.8 to 43.1 °C. The fastest increase in temperature was observed in the mice injected with MPG NPs. The temperature of the tumor site increased from 35.7 to 44.7 °C, which also proved that MPG NPs demonstrate good targeting of MDR gastric cancer cells, allowing more MPG NPs to gather at the tumor sites to exert the better PTT effect. All of the above results indicate that MPG NPs have a good in vitro and in vivo photothermal effect and can release heat under laser irradiation after being internalized by the cells, thus causing cell death (Fig. 5H).

Cellular uptake and affinity analysis

To verify whether MPG NPs can be used for in vivo MDR monitoring, cell uptake and affinity analyses were first performed. SGC 7901 ADR cells were used for internalization analysis, and the cell affinity of MPG NPs was verified in SGC 7901 ADR and SGC 7901 cell lines. The same concentration of MPG NPs was incubated with SGC 7901 ADR cells for different times, and the fluorescence intensity was observed by confocal microscopy. As shown in Additional file 1: Fig. S3A, blue represents the fluorescence of DAPI, and green represents the fluorescence of MPG NPs. The results showed that with increasing incubation time, the green fluorescence gradually increased, and the green fluorescence was mainly distributed in the cytoplasm, which indicated that MPG NPs were gradually internalized into the cytoplasm. The quantification of fluorescence intensity in Additional file 1: Fig. S3C also shows the same result. A cell affinity experiment was used to verify the specific targeting ability of MPG NPs on MDR gastric cancer cells. As shown in Additional file 1: Fig. S3B, in SGC 7901 ADR cells, the green fluorescence intensity after blocking with GMBP1 was significantly lower than the fluorescence intensity after MPG NP incubation. In SGC 7901 cells, the green fluorescence intensity after blocking with GMBP1 was not significantly different from the fluorescence intensity directly incubated with MPG NPs. The above results indicate that GMBP1 has the ability to specifically target SGC 7901 ADR cells because GMBP1 can specifically bind to the GRP78 receptor overexpressed on the surface of MDR cells [5]. The quantitative analysis shown in Additional file 1: Fig. S3D also produced the same results. The good specific targeting of MPG NPs to tumor cells also leads to less accumulation of MPG NPs in normal cells, thereby further reducing its in vivo toxicity. MPG NPs provide the basis for in vivo MDR monitoring and synergistic therapy with their specific tumor targeting.

Cellular inhibitory effect of MPG NPs

The toxicity and the inhibitory effect of nanomaterials on normal cells and tumor cells were evaluated by the MTT method, which is very important for MDR gastric cancer therapy. As shown in Fig. 6A, the cytotoxicity of MPG NPs to normal cells was concentration-dependent. With increasing MPG NP concentration, the survival rate of HUVECs decreased gradually. In previous studies, cell survival was still close to 60% when the concentration of Mn3O4@PEG was up to 40 µg mL− 1 [7]. The coating of PDA significantly reduced the toxicity of Mn3O4, and when the concentration reached 243 µg mL− 1, the survival rate of normal cells after Mn3O4@PDA NPs treatment was still above 80%. In SGC 7901 cells, MPG NPs did not show a significant inhibitory effect on the cells (Fig. 6B), while in SGC 7901 ADR cells, MPG NPs had a significantly enhanced inhibitory effect on the cells (Fig. 6C). The results show that low concentrations of MPG NPs will not cause significant damage to normal cells, and the presence of GMBP1 reduces the cytotoxicity caused by the tetrazolium compound T. Moreover, the high uptake of MPG NPs by SGC 7901 ADR cells also increased the inhibitory effect of MPG NPs on MDR gastric cancer cells, which was attributed to the presence of GMBP1 on the surface of MPG NPs. The survival rate of the cells after laser irradiation was measured in SGC 7901 and SGC 7901 ADR cells. As shown in Fig. 6D and E, laser irradiation did not cause significant cell death in the absence of MPG NPs. With increasing MPG NP concentration, the cell survival rate decreased significantly. When the concentration of MPG NPs reached 100 µg mL− 1, only a few cells survived, and under laser irradiation, the inhibitory effect of MPG NPs on SGC 7901 ADR cells was significantly stronger than that of SGC 7901 cells.

Fig. 6
figure 6

Tumor cell inhibitory effect of MPG NPs. A Survival rate of HUVECs after incubation with MPG NPs. B Survival rate of SGC 7901 cells after incubation with MPG NPs. C Survival rate of SGC 7901 ADR cells after incubation with MPG NPs. D Survival rate of SGC 7901 cells incubated with MPG NPs after laser irradiation. E Survival rate of SGC 7901 ADR cells incubated with MPG NPs after laser irradiation. F Clone images of cells incubated with MPG NPs after laser irradiation. G Quantification of the number of clones in SGC 7901 ADR cells. H Quantification of the number of clones in SGC 7901 cells. I Flow cytometry of cells incubated with different concentrations of MPG NPs with or without laser irradiation. J Staining image of cells with or without laser irradiation after incubation with MPG NPs

The inhibitory effect of MPG NPs on tumor cells was verified by colony formation assay. Images of cell clones treated with different concentrations of MPG NPs were obtained, and the number of colonies was quantified. As shown in Fig. 6F, in SGC 7901 and SGC 7901 ADR cells, the number of cell clones in the control group was the largest. With increasing MPG NP concentration, the number of cell clones gradually decreased. Figure 6G and H show the quantification of the number of cell clones in each group. The results show that when the MPG NP concentration reaches 100 µg mL− 1, the number of cell clones is significantly reduced, which is a significant difference compared with the control group. The above results indicate that MPG NPs have a good photothermal effect and can inhibit the growth of tumor cells after 808 nm laser irradiation. With increasing MPG NP concentration, the photothermal effect is more obvious, and the tumor cell inhibitory effect is also more obvious.

The apoptosis of cells after incubation with different concentrations of MPG NPs was observed by flow cytometry. As shown in Fig. 6I, the cells that were not treated with MPG NPs did not show obvious apoptosis. Without laser irradiation, slight early apoptosis appeared gradually with increasing MPG NP concentration. However, after 808 nm laser irradiation for 2 min, the number of apoptotic cells increased with increasing MPG NP concentration. The results show that the photothermal effect of MPG NPs causes significant cell apoptosis, verifying the good photothermal effect and the potential of in vivo PTT of MPG NPs. The inhibitory effect of MPG NPs on SGC 7901 ADR cells was further verified by cell staining. FDA was used to stain living cells with green fluorescence, and PI was used to stain dead cells with red fluorescence. As shown in Fig. 6J, in the absence of MPG NPs, neither laser irradiation nor nonirradiation caused significant cell death. The low concentration of MPG NPs only caused a few cell deaths, which is due to the CDT effect of MPG NPs in the cells. At the same concentration of MPG NPs, the number of cell deaths increased greatly after laser irradiation, which was caused by the photothermal effect of MPG NPs after laser irradiation. All the above results indicated that MPG NPs had a significant inhibitory effect on tumor cells and had the potential for synergistic therapy in vivo.

In vivo T1-weighted MRI for MDR monitoring

In vivo MDR monitoring is very important for the individualized treatment of gastric cancer. MRI was performed to verify whether MPG NPs can be used for in vivo MDR monitoring. An orthotopic gastric cancer mouse model was established, MPG NPs were injected in vivo, and MRI signal changes were monitored for 5 h. As shown in Fig. 7A, no significant signal enhancement at the tumor site was observed in the control group. The tumor site has been marked with a circle. The MRI signal intensity of the mice in the SGC 7901 ADR group increased significantly 2 h after the injection, and the MRI signal intensity was strongest at 3 h after the injection. In the SGC 7901 group, the MRI signal intensity also increased significantly after injection, but the signal intensity was weaker than that of the SGC 7901 ADR group. In the blocking group, GMBP1 was injected first, and then MPG NPs were injected half an hour later, the MRI signal intensity did not increase significantly. This is because free GMBP1 competes with MPG NPs to bind to the GRP78 receptors on the surface of MDR gastric cancer cells, so MPG NPs cannot be enriched at the tumor site, thus showing lower MRI signal intensity. It can also be seen from the quantitative results that in SGC 7901 ADR model mice, the MRI signal increased significantly and reached the maximum signal value 3 h after injection (Additional file 1: Fig. S3E). Major organs and tumor tissues of each group of mice were obtained, and immunofluorescence staining was performed. Figure 7B shows the staining images of each group of mice. The results showed that the fluorescence intensity of MPG NPs in the SGC 7901 ADR mouse model was significantly higher than that in the SGC 7901 mouse model and GMBP1 blocking group. This was attributed to the presence of the specific ligand GMBP1 on the surface of MPG NPs, which allowed more MPG NPs to enter SGC ADR cells. This is consistent with the results of in vivo MRI. These results indicate that GMBP1 can be used as a specific peptide to monitor MDR in gastric cancer.

Fig. 7
figure 7

In vivo MRI and immunofluorescence staining. A In vivo MRI images of mice in each group. B Immunofluorescence staining images of the main organs and tumor tissue sections of mice in each group

In vivo synergistic therapy for orthotopic MDR gastric cancer

The development of MDR is closely related to the TME, and tumor treatment strategies for the TME have been widely used to combat MDR. In vivo MRI has achieved accurate monitoring of MDR. The results of in vitro experiments have proven that MPG NPs can consume GSH and convert the endogenous H2O2 of the cell into highly toxic ·OH, thereby exerting the CDT effect. The good photothermal effect of Mn3O4 NPs can realize in vivo PTT, which can further enhance the CDT effect. To verify whether MPG NPs can be used for in vivo CDT/PTT synergistic therapy, an orthotopic MDR gastric cancer mouse model was established. As shown in Fig. 8A, the tumorigenesis of model mice was monitored by bioluminescence imaging, and synergistic therapy was performed for 20 days when the tumor size increased to an appropriate size. 6 h after the injection, the tumor sites of the mice in the PTT group were irradiated with an 808 nm laser. The bioluminescence imaging images of the mice were obtained 12 h after irradiation, and the weight changes of the mice were recorded. Figure 8B shows the simple principle of synergistic therapy. MPG NPs gather at the tumor site with abundant blood vessels through the EPR effect and the specific targeting effect of GMBP1 along with the blood circulation. Under irradiation with an 808 nm laser, Mn3O4@PDA NPs convert light energy into heat energy and locally release heat, thereby causing tumor cell apoptosis. The released Mn3O4 NPs react with GSH in tumor cells to generate Mn2+. Mn2+ and H2O2 in tumor cells mediate the Fenton-like reaction under HCO3 conditions to produce highly toxic ·OH, thereby killing tumor cells. The consumption of GSH by Mn3O4 also inhibits the elimination of ·OH by GSH to achieve a better CDT effect.

Fig. 8
figure 8

In vivo synergistic therapy. A synergistic therapy strategy for 20 days. B Schematic diagram of in vivo synergistic therapy. C Bioluminescence images of mice in each group within 20 days. D Photos of tumors in each group of mice. E H&E staining and TUNEL staining images of tumor sections of mice in each group

Figure 8C shows the bioluminescence images of each group of mice after 20 days of treatment. In the PBS group, the signal at the tumor site gradually increased, indicating that the tumor gradually became larger without treatment. After PBS injection and 808 nm laser irradiation, the signal at the tumor site also showed a gradual increase, but the signal was weaker than that in the PBS group. This indicates that laser irradiation alone can slow down the growth of the tumor to a certain extent, but the therapeutic effect is not better because of the low temperature. The bioluminescence signal of the mice injected with Mn3O4@PDA NPs was weaker, but the tumor was not suppressed. This is because the injection of Mn3O4@PDA NPs alone can only have a tumor treatment effect through CDT. The tumors of mice injected with MPG NPs became significantly smaller because the surface of GMBP1 gave MPG NPs better tumor targeting ability, causing more MPG NPs to accumulate in the tumor site to achieve a better CDT effect. After the injection of MPG NPs and laser irradiation, the tumor bioluminescence signal of mice was significantly reduced, the tumor gradually decreased, and the tumor disappeared finally. This is because the Mn3O4 NPs exert a good photothermal effect under laser irradiation. Surface-wrapped PDA also enhances the PTT effect and combines with the CDT effect of MPG NPs to achieve a better synergistic therapy effect. The quantitative signal intensity of bioluminescence is shown in Additional file 1: Fig. S4A–E. Figure 8D shows a picture of the tumor after anatomy. The tumors in the MPG NPs + laser group were the smallest, and among the 5 experimental mice, the tumors in 2 mice disappeared and recovered completely. The quantitative results of tumor weight in Additional file 1: Fig. S4F also showed the same results. The tumor weight of the MPG NPs + laser group was significantly different from that of the other groups. During the treatment period, the body weight changes of the mice in each group were monitored. As shown in Additional file 1: Fig. S4G, the mice in the PBS group were weakened due to the gradual enlargement of tumors, which led to significant weight loss. The weight of the mice in the MPG NPs + laser group also gradually increased due to the recovery of their physical condition. These results indicate that MPG NPs have a good synergistic therapeutic effect. The tumors of each group of mice were sectioned and stained for observation. As shown in Fig. 8E, the H&E staining results showed that the tumor cell structure of the mice in the MPG NPs + laser group was destroyed and showed a necrotic form. The results of TUNEL staining (Fig. 8E and Additional file 1: Fig. S4H) showed that the tumor apoptosis of mice in the MPG NPs + laser group was the most severe, which was consistent with the results of in vivo synergistic therapy. All of the above results show that MPG NPs have good synergistic therapy effect and can be used for CDT/PTT synergistic therapy for gastric cancer MDR.

In vivo biosafety assessment

To investigate the in vivo toxicity of MPG NPs, acute toxicity and hemolysis tests were carried out. Additional file 1: Fig. S5A shows the hemolysis rate of various materials. Among them, the hemolysis rate of Mn3O4@PEG NPs is approximately 10%, which indicates certain blood toxicity. The hemolysis rates of MPG NPs, PDA and GMBP1 were 1.8%, 2% and 0.7%, respectively. The results show that they are almost nontoxic, further indicating that MPG NPs can be used for in vivo studies. Then, an in vivo acute toxicity test was carried out. Three groups of healthy mice were injected with GMBP1, Mn3O4@PDA NPs and MPG NPs at a concentration of 50 mg kg-1. The survival and body weight changes of the mice within 14 days were observed. The results showed that all 30 mice survived after 14 days. The body weight of each group of mice shown in Additional file 1: Fig. S5B exhibits a steady upward trend. The pathological changes of mice in each group were recorded within 14 days. As shown in Additional file 1: Fig. S5C, the mice in each group showed pathological changes to a certain extent, such as coarse hair, bulgy eyeballs, and bradykinesia, but this situation was improved after a week, this may be due to the toxicity of high concentrations of manganese ions. The specific statistical results of the pathological characteristics of mice in each group are shown in Additional file 1: Table S1. Among them, the pathological characteristics of mice in the Mn3O4@PDA NP treatment group were more obvious than those in the GMBP1 and MPG NP treatment groups. The pathological section in Additional file 1: Fig. S5D shows that GMBP1, Mn3O4@PDA NPs and MPG NPs exerted no obvious damage on the heart, liver, or kidneys of mice. This is due to the good tumor cell targeting of MPG NPs, which allows them to accumulate more in the tumor site, avoiding damage to normal tissues. The above results all indicate that MPG NPs have good biosafety.

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