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All-in-one approaches for triple-negative breast cancer therapy: metal-phenolic nanoplatform for MR imaging-guided combinational therapy | Journal of Nanobiotechnology

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Preparation and optimization of nanoparticles

Even though EGCG and Fe3+ ions have been widely used to prepare MPNs, there were rare reports about using MPNs as carriers for delivery of BLM. Owing to the presence of metal-binding domain in BLM, Fe3+ ions could assemble with BLM in water, forming the core of nanoparticles [38, 39]. Then redundant Fe3+ ions further coordinated with EGCG at the interface of ethanol/water, composing MPNs (Fig. 1A and Additional file 1: Figure S1). We firstly investigated the proportion of three components required for the formation of BFE NPs. At beginning, the proportion of EGCG was explored by varying its input with fixed amount of BLM and Fe3+. As shown in Fig. 1B and Additional file 1: Figure S2A, when the molar ratio between BLM, EGCG and Fe3+ was set at 1:3:6, the corresponding nanoparticles exhibited the darkest color, whereas the particle size reached minimum and the zeta potential also decreased to the lowest. Similarly, the influence of the amount of Fe3+ on BFE NPs was examined. Using particle size and zeta potential as indicators, it has been further confirmed that the prepared nanoparticles were most suitable when the proportion of three components was 1:3:6 (Fig. 1C and Additional file 1: Figure S2B). Therefore, the molar ratio between BLM, EGCG and Fe3+ in the formation of BFE NPs was fixed at 1:3:6 in the following investigation.

Fig. 1
figure 1

A Schematic description of BFE NPs preparation. The particle size and zeta potential of BFE NPs at different molar ratios of EGCG (B) and Fe3+ (C). The particle size, PDI (D) and photograph (E) of BFE NPs in various media. F The long-term stability of BFE NPs. G Schematic description of BFE@BSA NPs preparation. H The particle size and PDI of nanoparticles at different BSA concentrations. I The long-term stability of BFE@BSA NPs. J The summary of the four types of nanoparticles. The TEM images of BFE NPs (K) and BFE@BSA NPs (L). Scale bars were 200 nm

The stability of nanoparticles is a prerequisite for subsequent application and storage. In order to simulate different biological conditions, the particle size changes of BFE NPs incubated in different media (H2O, Glu, NS, PB, and PBS) were monitored. After 24 h, BFE NPs were quite stable in both water and glucose solution, as the particle size of BFE NPs remained unchanged (Fig. 1D and E). But the particle size of BFE NPs increased exponentially in NS, PB and PBS, and sedimentation was even observed in PB and PBS. Furthermore, there was no precipitation of BFE NPs in 10 or 50% FBS-M or 100% FBS, and the particle size of nanoparticles slightly increased and then stayed steady (Additional file 1: Figure S3). Thus, we hypothesized that BFE NPs might adsorb proteins from the serum, which facilitated the stability of nanoparticles. In long-term stability study, BFE NPs were steady and remained the similar size for 7 days in the complete medium containing 10% FBS (Fig. 1F and Additional file 1: Figure S4A). In brief, BFE NPs aggregated in various salt solutions, but they exhibited colloidal stability in serum-containing solution, indicating that serum proteins might protect BFE NPs from aggregation.

Polyphenols are highly adherent and exhibit multiple interactions (e.g., hydrogen bonds, hydrophobic, and electrostatic interactions), which allow them to form robust conjugates with other substances. BSA has been widely used to modify nanoparticles to improve the stability. In order to improve the stability of BFE NPs, BFE NPs were further modified with BSA by incubation in BSA solution through surface adsorption (Fig. 1G). When the concentration of BSA reached 5 mg/mL (Fig. 1H), the nanoparticles could remain stable in PBS. Subsequently, with the concentration of BSA continually increased, the particle size of nanoparticles gradually increased. Therefore, the BSA concentration of 5 mg/mL was selected to prepare BFE@BSA NPs. As expected, the particle size of BFE@BSA NPs remained constant for 7 days in all media, including H2O, PBS and 10% FBS-M (Fig. 1I and Additional file 1: Figure S4B). The introduction of BSA endowed nanoparticles with great physiological stability and long-term stability, which laid a sufficient foundation for subsequent application.

To sum up, BLM + Fe3+ or BLM + EGCG could form nanoparticles (BF NPs or BE NPs). But only when three components were all present, nanoparticles with desired structure could be fabricated. As shown in Fig. 1J, BFE NPs which were prepared in optimal proportion displayed the particle size of 158.9 ± 1.8 nm and the zeta potential of − 38.1 ± 3.5 mV and presented a dark black appearance. The negative surface charge of BFE NPs indicated the structure of MPNs. After BSA coating on the surface of nanoparticles, BFE@BSA NPs remained the black appearance, while their particle size increased by ~ 15 nm (173.0 ± 1.4 nm) than BFE NPs and zeta potential was back to − 11.5 ± 1.1 mV. As a result of the masking of BSA, BFE@BSA NPs were less charged, which remarkably improved the stability of nanoparticles.

Characterization and spectra of nanoparticles

The morphology of nanoparticles was studied by TEM. As shown in Fig. 1K–L and Additional file 1: Figure S5, BFE NPs possessed network-like structures, which indicated the successful formation of MPNs. While the images of BFE@BSA NPs presented scattered particles because BSA facilitated the dispersion of nanoparticles. As shown in Additional file 1: Table S1, the EE of BLM and Fe3+ in BFE@BSA NPs was 65.0% and 77.2% (80.4% and 94.9% in BFE NPs), respectively, indicating this delivery system realized efficient drug packaging. In order to confirm the formation of nanoparticles, the UV–vis spectra, fluorescent spectra and FT-IR spectra were subsequently applied to analyze the formation mechanism. In Fig. 2A, the maximum absorption wavelength of BLM at 291 nm was red-shifted to 297 nm in the UV–vis spectrum of BFE@BSA NPs, which manifested the π-π interactions between BLM, EGCG and BSA. Furthermore, BFE@BSA NPs and direct mixture of EGCG + Fe3+ exhibited broad absorption in the NIR window, confirming that Fe3+-based MPNs had the ability to serve as photothermal reagents. In Fig. 2B, BLM exhibited a fluorescence emission from 330 to 480 nm with excitation wavelength at 308 nm in its fluorescent spectrum. However, the fluorescence of BLM was quenched in BFE@BSA NPs as a result of the formation of coordination bonds between EGCG and Fe3+. In Fig. 2C, the -OH stretching bands (3556, 3479 and 3357 cm−1) of pure EGCG were not observed in the FT-IR spectrum of BFE@BSA NPs, demonstrating the formation of coordination bonds. BFE@BSA NPs exhibited the characteristic amide I and amide II band vibrations (1648 and 1550 cm−1) of BLM and BSA, indicating the existence of BLM and BSA in nanoparticles. All of the above results proved the successful preparation of BFE@BSA NPs.

Fig. 2
figure 2

The UV–vis spectra (A), fluorescent spectra (B) and FT-IR spectra (C) analysis. D The photographs of BFE NPs and BFE@BSA NPs dissociation by NaCl, Urea, EDTA and SDS. E The fluorescence recovery analysis of BFE@BSA NPs after treatment with EDTA at different time points. F The changes of particle size of BFE@BSA NPs at different pH conditions over 72 h. The fluorescence recovery analysis of BFE@BSA NPs in PBS at pH 6.5 (G) and pH 5.5 (H). I The drug release behavior of BLM in PBS at different pH value. J The temperature elevation of BFE@BSA NPs under 808 nm laser irradiation with different power density for 10 min at Fe3+ concentration of 0.8 mM. K The temperature elevation of BFE@BSA NPs under laser irradiation (808 nm, 2.5 W/cm2) for 10 min at different Fe3+ concentrations. (Note: The trend of 0.8 mM is the same one of 2.5 W/cm2 in Fig 2J) L The temperature changes of BFE@BSA NPs within 5 laser-on and off cycles. Statistical p values: *p < 0.05

Self-assembly mechanism study of BFE NPs and BFE@BSA NPs

In order to further confirm the self-assembly mechanism of BFE NPs and BFE@BSA NPs, the prepared nanoparticles were dispersed into different solutions (NaCl, urea, EDTA and SDS) to determine the role of electrostatic force, hydrogen bonds, coordination bonds and hydrophobic force, respectively. In Fig. 2D, when BFE NPs were dispersed into 0.9% NaCl solution (electrostatic force-eliminating agents), a small amount of precipitation was observed, indicating there were certain electrostatic interactions within nanomedicines. Changes and precipitation were more obvious when BFE was mixed with urea solution which could deconstruct hydrogen bonds, suggesting the hydrogen bonds were involved in the formation of BFE NPs. However, it was found that SDS did not affect nanoparticles, suggesting hydrophobic force barely existed during the assembly process of BFE NPs. The most obvious change of BFE NPs was observed in appearance, where precipitate formed immediately, upon mixing with EDTA solution (coordination bonds-eliminating agents). These results implied that EDTA was the most effective treatment to disintegrate the BFE NPs and coordination force played the main force in the formation of BFE.

To sum up, we could speculate different forces that participated in the formation of nanoparticles as follow: coordination bonds > hydrogen bonds > electrostatic force  hydrophobic force. Furthermore, when the BFE@BSA NPs were placed in the above solution, the changes were smaller than that of BFE NPs in appearance. This indicated that the presence of BSA could improve the stability of nanoparticles to a certain degree. It has been proved that the fluorescence of BLM in BFE@BSA NPs was quenched, owing to coordination between BLM and Fe3+ (Fig. 2B). Therefore, we further investigated the fluorescence recovery ability of BLM from BFE@BSA NPs by adding EDTA solution. The degradation of BFE@BSA NPs was supposed to induce the release of BLM, leading to fluorescence recovery. As shown in Fig. 2E, upon adding EDTA, the fluorescence of BLM gradually recovered and fluorescence intensity gradually increased with incubation time. It indicated that EDTA could destroy the structures of BFE@BSA NPs and lead to the release of BLM. It further confirmed that coordination force played a vital role in the self-assembly process of nanoparticles.

In vitro pH sensitive study

The BFE@BSA NPs was designed with the characteristics of pH-responsive degradation in an acidic environment, owing to the pH responsiveness of coordination force and hydrogen bonding. In acidic conditions, the disassemble of BFE@BSA NPs was accompanied by BLM release in the acidic TME. To evaluate the pH-responsive capability of BFE@BSA NPs, the size distribution of nanomedicines was measured firstly through dispersing them into buffer solution with pH at 6.5 and 5.5. From the results in Fig. 2F, with the extension of incubation time, the particle size of nanoparticles increased gradually in an acidic environment. The particle size of BFE@BSA NPs remained steady with negligible changes at pH 7.4. But at pH 6.5 and 5.5, BFE@BSA NPs became larger than before, indicating that the nanoparticles were pH-responsive.

Furthermore, the fluorescence recovery ability of BLM from BFE@BSA NPs under acidic conditions (pH 6.5 and 5.5) was also investigated. As expected, the fluorescence intensity of BLM increased gradually with incubation time at both pH values in Fig. 2G and H. Moreover, the fluorescence intensity of BLM recovered more quickly at pH 5.5 than that at pH 6.5. These results implied that BFE@BSA NPs could gradually dissociate and release the BLM in an acidic environment.

In vitro drug release

To accurately access the BLM release behavior from BFE@BSA NPs in the physiological condition, tumor sites and intracellular acidic environment, in vitro drug release tests were performed in PBS solutions with pH 7.4, pH 6.5 and pH 5.5, to simulate certain conditions. As shown in Fig. 2I, the amount of BLM released from BFE@BSA NPs was gradually increased as the pH of releasing medium decreased from 7.4 to 5.5. At pH 7.4, the amount of BLM released within the entire experimental period (72 h) was around 30%, while BLM released was up to 43% in PBS solution at pH 5.5. The pH sensitive release profile was mainly ascribed to the dissociation of the coordination bands in BFE@BSA NPs in acidic environment.

In vitro photothermal evaluation

In our previous study, it was found that Fe3+ ions-based MPNs exhibited a broad absorption in the NIR region, which indicated that the designed reagents might have a photothermal transduction effect. To test the photothermal conversion efficiency of BFE@BSA NPs, the temperature differences (ΔT) of BFE@BSA NPs solution was checked under different Fe3+ concentrations or power densities. In Fig. 2J, K and Additional file 1: Figure S6A, B, with the increase of laser intensity (from 2.0 to 3.0 W/cm2) or iron ions concentration (from 0.2 to 0.8 mM), the temperature of BFE@BSA NPs solution increased significantly. The temperature of BFE@BSA NPs with Fe3+ concentration at 0.8 mM could increase up to 48.1 ℃ with temperature change about 21 ℃ after irradiation for 10 min (808 nm laser at 2.5 W/cm2). These results suggested that BFE@BSA NPs could serve as effective PTAs.

To further investigate the photothermal stability of nanomedicines, BFE NPs and BFE@BSA NPs (0.8 mM) were irradiated for 5 cycles of laser on (808 nm laser at 2.5 W/cm2, 10 min) and laser off (10 min), respectively. After 5 cycles, the ΔT between adjacent peaks was within 0.5 ℃ according to Fig. 2L. It indicated the excellent photothermal stability of both BFE NPs and BFE@BSA NPs under repeated NIR laser irradiation, while coating with BSA did not affect the photothermal conversion efficiency of nanoparticles.

In vitro cytotoxicity assay

The cell viability was firstly determined by MTT assay to investigate the in vitro anti-tumor ability of BFE@BSA NPs. 4T1 cells were incubated with free BLM or BFE@BSA NPs at different BLM concentrations. As shown in Fig. 3A, both free BLM and BFE@BSA NPs showed weak cytotoxicity at low concentrations. The cell viability of BFE@BSA NPs at BLM concentration of 8 μM was 53.2%, which was lower than that of free BLM (66.5%). And the cell viability of BFE@BSA NPs + L (BLM: 8 μM) was 47.1%, indicating that BFE@BSA NPs could efficiently kill tumor cells with laser irradiation. Then, live/dead staining assay was conducted by staining cells with Calcein-AM and PI after respective treatments. Calcein-AM (green) could easily penetrate live cell membrane to mark live cells, while PI (red) could only reach the cell nucleus through the disordered membrane of dead cell. As shown in Fig. 3B, the cells which were treated with laser alone showed almost no apparent red fluorescence. More red fluorescent signals were found in the groups treated with free BLM or BFE@BSA NPs (BLM: 8 μM), and cells treated with BFE@BSA NPs + L presented obvious red fluorescence. All the results illustrated the synergistic cytotoxicity of combination therapy.

Fig. 3
figure 3

A The viability of 4T1 cells after different treatments (n = 6). B 4T1 cells stained by Calcein-AM/PI kit after different treatments. Scale bars were 20 μm. C The cellular uptake of coumarin-6 labeled BFE NPs and BFE@BSA NPs. Scale bars were 20 μm. D Schematic illustration of intracellular mechanisms. E The flow cytometry analysis of ROS generation after different treatments and (F) corresponding MFI values (n = 3). G The fluorescence images of ROS probes after different treatments. Scale bars were 50 μm. Statistical p values verse the control group: *p < 0.05, **p < 0.01, ***p < 0.001

In vitro cellular uptake study

It was reported that the cellular uptake of nanomedicines was significantly influenced by their surface physicochemical characteristics. In order to understand whether the presence of BSA in our system would facilitate the cellular uptake, we evaluated the effect of BSA modification on the in vitro cellular uptake. Before that, green fluorescent dye, coumarin-6, was loaded into BFE NPs during the preparation for fluorescent observations. The internalization of BFE NPs and BFE@BSA NPs into 4T1 cells was observed by fluorescence microscopy. The blue area was the nuclei of 4T1 cells stained by DAPI and the green fluorescence resulted from coumarin-6 loaded nanoparticles. As shown in Fig. 3C, the amount of phagocytosed nanoparticles, no matter BFE NPs or BFE@BSA NPs, both increased with time, as stronger green fluorescence was observed at 6 h than that at 2 h, indicating the internalization of nanoparticles occurred in a time-dependent manner. What’s more, after incubation with BFE NPs or BFE@BSA NPs, the cytoplasm of 4T1 cells presented different intensities of coumarin-6 fluorescence. The fluorescence of cells treated with BFE@BSA NPs was much stronger than that in BFE NPs group, indicating that the surface modification of BSA could promote cellular uptake of nanoparticles.

In vitro ROS generation

It is a truism that higher than the physiological level of ROS would trigger the damage of proteins, organelles and nucleic acids, thus leading to cell apoptosis. Excessive production of ROS within cancer cells, such as superoxide anion radical (O2) and hydroxyl radical (•OH), is often considered to be a significant condition for killing tumor cells. The designed system was supposed to produce excess ROS in multiple directions (Fig. 3D). In combination with FeII, BLM was transformed into an activated form and could convert oxygen into hydrogen peroxide (H2O2). Highly toxic hydroxyl radicals were produced by the Fenton reactions under the high concentration of H2O2, leading to ROS amplification. Moreover, the transformation between Fe3+ ions and Fe2+ ions consumed glutathione heavily. This kind of GSH depletion could protect generated ROS from scavenging. Elevated temperature caused by PTT could further promote the production of ROS. Therefore, we evaluated the ROS generation ability of BFE@BSA NPs by incubation with 4T1 cancer cells. According to the fluorescence changes of a ROS-sensitive probe, 2′,7′-dichlorofluorescin diacetate (DCFH-DA), the amount of generated ROS could be confirmed. As shown in Fig. 3G, weak green fluorescence (DCF) was observed in control group or cells treated with laser, indicating that ROS produced by the metabolism of cells themselves was limited. The ROS level in 4T1 cancer cells treated with BLM alone increased slightly. It was reasonable since BLM could efficiently promote the formation of hydrogen peroxide only when catalyzed by adequate iron ions. Treating 4T1 cells with BFE@BSA NPs could facilitate the generation of ROS, as an increased fluorescence signal was observed in the cytoplasm. Upon laser irradiation, bright fluorescence was observed, indicating that the significantly enhanced ROS amplification effect was triggered by NIR. Furthermore, similar results were mirrored by flow cytometry analysis which were presented in Fig. 3E and F. These results implied that BFE@BSA NPs combined with PTT could synergistically induce ROS amplification.

In vitro MRI study of BFE@BSA NPs

The incorporation of Fe3+ ions enbaled BFE@BSA NPs to be potential MRI contrast agents. To further evaluate the contrast efficacy of BFE@BSA NPs, their longitudinal relaxivity (r1) and transverse relaxivity (r2) were tested firstly by scanning BFE@BSA NPs with different concentration gradients of Fe (0, 0.1, 0.2, 0.3, 0.4, and 0.5 mM). According to the graph data (Fig. 4A and Additional file 1: Figure S7), BFE@BSA NPs exhibited the r1 value of 0.96 mM−1 s−1 in water. The r2 value of BFE@BSA NPs was determined to be 2.41 mM−1 s−1, which was too low for BFE@BSA NPs to perform as T2-weighted contrast agents. But r2/r1 = 2.51 < 3, indicating BFE@BSA NPs were potential T1-weighted contrast agents for further application. [49] In Fig. 4B, it showed the T1-weighted images of BFE@BSA NPs at different Fe3+ concentrations, which presented positive bright contrast increasing. The MR signal was enhanced linearly with the increasing concentrations of Fe3+. BFE@BSA NPs dispersed in agarose gel also showed the obvious results (Additional file 1: Figure S8). The T1 value was measured by the T1 mapping images. As shown in Fig. C, BFE@BSA NPs could significantly shorten T1 relaxation time both in water and agarose gel, indicating they have good MR imaging ability in vitro. Then, BFE@BSA NPs were used for cellular MR imaging. [50] After incubation with nanoparticles for 4 h, the T1 value of 4T1 cells treated with BFE@BSA NPs was found to be significantly lower than that of cells without nanoparticles (Fig. 4D, E), indicating BFE@BSA NPs had the potential for further in vivo MR imaging.

Fig. 4
figure 4

A The linear fitting of 1/T1 of BFE@BSA NPs at different Fe3+ concentrations. The T1-weighted MR images (B) and T1 mapping images (C) of BFE@BSA NPs nanoparticle in vitro. D The T1 WI and T1 Map of BFE@BSA NPs nanoparticle for cellular imaging. E The T1 value of each group (n = 3). F Schematic description of in vivo MR scanning plan. G The axial T1 WI of mice at different time points. The quantitative analysis of relative T1 signal (H) and SNR (I) in tumor region (n = 6, the same mouse for continuous monitoring). J The coronal T1 WI and T1 Map of mice at different time points. The quantitative analysis of SNR (K) and T1 value (L) in tumor region (n = 3, different mice for each time point). F The in vivo biodistribution of the DiR-loaded BFE@BSA NPs (n = 3). G The quantitative analysis of fluorescence intensity in tumor

In vivo MR imaging of mice

It is extremely important to accurately monitor the size and location of tumor in the course of tumor therapy. Especially for photothermal or photodynamic therapy, accurate imaging of tumors can guide the course of treatment, monitor therapeutic efficacy, and reduce unexpected damage to normal tissues. MRI has become a diagnostic and research tool in treating various tumors because of its ability of accurately delineating the detailed images of the tumor tissue. As shown in Fig. 4F, 4T1 tumor-bearing BALB/c mice were i.v. injected with BFE@BSA NPs to assess in vivo MR imaging ability. In Fig. 4G, compared to pre-injection, the tumor region of mice was significantly brighter after injection of BFE@BSA NPs, owing to the effective accumulation of BFE@BSA NPs at the tumor site. The contrast between the tumor tissue and surrounding normal tissue was more pronounced, making tumor boundary clearer. In Fig. 4H, BFE@BSA NPs was continuously enriched at the tumor site and the T1 MR signal intensity gradually increased. At 4 h post-injection, the relative T1 signal of tumor site reached the maximum, which was about 1.63 times higher than that of pre-injection. After that, the MR signal intensity gradually decreased, or even fell below the value of pre-injection since 12 h post-injection. The quantitative analysis of SNR in tumor region presented the similar results (Fig.4I). It might be caused by the increased tumor necrotic areas (red arrow represented the necrosis of tumor tissue), which indicated the cytotoxic effect of BFE@BSA NPs upon arrival of the tumor region. [51] From all these results, it could reflect that effective accumulation of BFE@BSA NPs at the tumor sites could favor the precise MR imaging and cause necrosis of the tumor tissue, facilitating further therapeutic applications.

Based on the above axial images, coronal MR scanning was performed to study various organs of the mice. Furthermore, the coronal T1-weighted MR images and T1 mapping images of mice showed the similar trend. As shown in Fig. 4J, after intravenous injection of BFE@BSA NPs, the tumor became brighter over time compared to other organs, and the average SNR of tumor gradually increased (Fig. 4K). In T1 mapping images, BFE@BSA NPs visibly decreased the T1 value of tumor, the T1 relaxation time was gradually shortened (Fig. 4L). In Additional file 1: Figure S9, the tumor and other organs could be clearly distinguished (the organs were marked with different colored arrows), and the SNR of different organs increase over time. Although the SNR of different organs increased over time, the relative signal ratio of tumor to other organs also increased, indicating that BFE@BSA NPs exhibited good enhancement effects on the signal of tumor.

Biodistribution study of BFE@BSA NPs

To evaluate the biodistribution of BFE@BSA NPs within the body, 4T1 tumor-bearing BALB/c mice were established and tested. Fluorescent dye DiR labeled BFE@BSA NPs or free DiR were injected into tumor-bearing BALB/c mice, respectively. The mice were sacrificed to obtain tumors and main organs at designed time intervals (8, 12 and 24 h). As shown in Fig. 5A, quite weak DiR fluorescence intensity was observed and remained the same level at all the time points in the tumor from mice intravenously injected with free DiR. It was similar trend as previous reports, which was ascribed to its weak tumor retention. [52] Moreover, the liver and spleen were the main accumulation organs of free DiR. By contrast, stronger fluorescence intensity was observed in the tumor from mice treated with BFE@BSA NPs. In Fig. 5B, quantification of fluorescence intensity indicated that the accumulative fluorescence intensity at the tumor site increased with prolonging time and reached the maximum fluorescence intensity at 12 h post-injection (Additional file 1: Figure S10). The quantitative results of relative fluorescent ratio of tumor/liver displayed the similar trend (Fig. 5C). [53] It was worth mention that the accumulation of BFE@BSA NPs in the liver and spleen was significantly reduced, indicating that our nanomedicine was able to prolong the circulation lifetime of drugs and change metabolic pathways of drugs in the body. These results implied that BFE@BSA NPs could accumulate in the tumor region effectively.

Fig. 5
figure 5

A The in vivo biodistribution of the DiR-loaded BFE@BSA NPs (n = 3, different mice for each time point). The quantitative analysis of fluorescence intensity of tumor (B) and relative fluorescent ratio of tumor/liver (C). D Schematic diagram of 4T1 tumor treatment. The 4T1 tumor growth curve (E), body weight of mice (F) and tumor weight (G) after different treatments (n = 5). H The photograph of tumors after different treatments. I H&E and masson’s trichrome staining of the lung sections from the mice after different treatments. Statistical p values: *p < 0.05, **p < 0.01, ***p < 0.001

In vivo dose exploration of BLM

Even though BLM is an effective glycopeptide anticancer drug that could affect the cutting of single- and double-stranded DNA, the risk of pulmonary fibrosis caused by BLM is still not negligible. In order to achieve the highest anti-tumor activity of BLM while minimizing its potential side effects, before applying BFE@BSA NPs into further in vivo anti-tumor experiment, the optimal dosage of BLM was firstly explored. As the experimental scheme diagram of treatment shown in Fig. 5D, 4T1 tumor-bearing BALB/c mice were i.v. injected with PBS or BFE@BSA NPs with different concentrations (BLM: 2.5, 5 and 10 mg/kg) on day 5, 8 and 11. Both the body weight and tumor volume were measured every 2 days and tumors from mice with various treatments were separated and weighed on day 21 post-injection. As shown in Fig. 5E–H and Additional file 1: Figure S11A, compared with PBS, all the groups treated with BFE@BSA NPs exhibited anti-tumor effect (inhibition rate of 47.0% for 2.5 mg/kg, 61.7% for 5 mg/kg and 67.7% for 10 mg/kg) due to effective killing effect of nanoparticles on tumors.

According to the tumor growth curve, mice treated with BFE@BSA NPs (BLM: 5 mg/kg or 10 mg/kg) exhibited similar tumor-suppressive power, as no significant difference was observed both in the tumor volume and tumor weight. But when compared to mice treated with 2.5 mg/kg, better tumor inhibition with significantly smaller tumor size and low tumor weight was found in groups treated with 5 or 10 mg/kg. Then, the safety of nanomedicine was monitored by analyzing the weight of the mice and lung section. It was found that 10 mg/kg of BLM treatment gave rise to the obvious loss of body weight, while the weight of mice treated with 5 mg/kg remained unchanged. It might mean that BFE@BSA NPs (BLM: 10 mg/kg) had overrun the safe dose. The idea was further confirmed in the results of lung section by histological analysis. As shown in Fig. 5I and Additional file 1: Figure S11B, the extensive inflammation was observed in the group of 10 mg/kg, and this was also associated with widespread collagen (blue area by Masson’s trichrome staining) accumulation and alveolar structure disorder. While the group of 2.5 and 5 mg/kg presented mild inflammation, and there were no obvious pathological changes in lung structures. BFE@BSA NPs (BLM: 10 mg/kg) showed more severe pulmonary fibrosis of mice than other groups, which represented dose-dependent side effects. Therefore, the concentration of nanomedicine was set to BFE@BSA NPs (BLM: 5 mg/kg) in the following experiments.

In vivo anti-tumor efficiency of synergistic therapy

To further evaluate the combined therapeutic effects of CDT and PTT, 4T1 tumor-bearing BALB/c mice were i.v. injected with PBS, free BLM (5 mg/kg) or BFE@BSA NPs (BLM: 5 mg/kg) three times on day 6, 9 and 12. Half of the mice that received BFE@BSA NPs were further locally irradiated under NIR laser every 12 h-post injection at a power density of 2.5 W/cm2 for 2 min (Fig. 6A). Both the body weight and tumor volume were measured every 2 days and tumors and main organs from mice with various treatments were separated and weighed on day 21. As recorded in Fig. 6B–D and F, BLM and BFE@BSA NPs treatment presented moderate tumor inhibitory effect comparing to PBS group. The tumor inhibitory rate of BLM and BFE@BSA NPs were 58.4% and 69.0%, respectively. The superior anti-tumor efficiency of BFE@BSA NPs than free BLM was attributed to the enhanced accumulation at tumor site and the ROS amplification effect of nanomedicine. Beyond expectation, the BFE@BSA NPs + L group showed the eminent inhibition compared with other groups (tumor inhibition rate was calculated to be 93.7%) without obvious weight loss of mice. In Fig. 6E and Additional file 1: Figure S12, complete tumor ablation was achieved in five mice of all eight mice (5/8). During the entire experimental period, the therapeutic efficacy was maintained well and no recurrence was observed in the ablated tumor. These results demonstrated the superiority of combination therapy of chemotherapy, CDT and PTT. Histological analysis of the lung sections stained with H&E and Masson’s trichrome was conducted to assess the side effects of pulmonary fibrosis. As shown in Fig. 6G and H, severe inflammation was not observed in each group, and there were no obvious pathological changes in the lungs of all groups. In general, BFE@BSA NPs + Laser could achieve the desirable therapeutic effect with minimal toxic side effects.

Fig. 6
figure 6

A Schematic diagram of 4T1 tumor treatment. The 4T1 tumor growth curve (B) tumor weight (C) and tumor inhibition rate (D) after different treatments (n = 8). E The photograph of tumors after different treatments. F The body weight of mice after different treatments. H&E (G) and masson’s (H) trichrome staining of the lung sections from the mice after different treatments. Statistical p values: *p < 0.05, **p < 0.01, ***p < 0.001

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