Development of in vitro and in vivo c-Met targeted dual-modal nanoprobes for NIR II fluorescent bioimaging and magnetic resonance imaging of breast carcinoma metastasis

2.1 Preparation of Fe₃O₄

The magnetic Fe₃O₄ nanocores were synthesized by the common solvo-thermal strategy (Liu, et al., 2020). Briefly, 2.7 g of FeCl₃·6H₂O, 7.2 g of sodium acetate were concurrently added into 100 mL ethylene glycol solution with vigorous magnetic stirring (1000 rpm/min). Above homogenous-yellow aqueous mixture was carefully transferred into a 100 mL of Teflon-lined stainless-steel autoclave which was sealed and maintained in an oven 200℃ for 8 hr. Subsequently, the autoclave cooled down at room temperature and the as-made black magnetic Fe₃O₄ nanoparticles were washed by anhydrous ethanol for 10-times and the prepared Fe-based samples were dried over-night at 60℃.

2.2 Synthesis of Fe₃O₄ @mSiO₂

Mesoporous silica coating was fabricated according to previous report work (Wei, et al., 2023). For silica based nanoshell wrapping, 100 mg of Fe₃O₄ nanoparticles were pre-treated with 50 mL, 0.1 mol/L of hydrochloric acid with a 10 min of continuous sonication. Then the hydrophilic magnetic nanocrystals were collected by a magnet and washed 3-times by deionized water. Thereafter, the prepared Fe-based nanoparticles were added in aqueous solution, containing 70 mL of ethanol, 80 mL of deionized water and 0.3 g of cetyltrimethyl ammonium bromide (CTAB). After vigorous magnetic stirring (450 rpm/min) for preparing a homogenous dispersion, 1.2 mL, 28 % ammonia was added and maintained stirring for 1 h. Latterly, 0.3 mL of tetraethyl orthosilicate was put into the above mixed solution and carried out a continuous magnetic stir for at 37 °C 12 hr. These core–shell magnetic nanoparticles Fe₃O₄@mSiO₂ with mesoporous shells were separated and washed sequentially by ethanol and deionized water for 6-times, then, the collected samples were re-dispersed in a 60 mL, 1 % NH₄NO₃ aqueous solution which was subjected to a sustained refluxing for 12 h. The final core–shell Fe₃O₄@mSiO₂ were obtained by a magnet, washed with anhydrous ethanol, and dried at 60 °C vacuum over-night.

2.3 Pegylation of Fe₃O₄@mSiO₂

Out-layer silica surface PEGylation was then performed. Typically, 50 mg of Fe₃O₄@mSiO₂ was added in 10 mL of aqueous solution containing 100 mg of PEG₂₀₀₀-NH₂. The mixture was gently stirred for 12 hr at room temperature, the amino group’s decorated Fe₃O₄@mSiO₂ nanocomposite (Fe₃O₄@mSiO₂-PEG) were successfully prepared. Immediately, residual PEG₂₀₀₀-NH₂ was removed by 3-times washing with water by a high-speed centrifugation (17000 rpm, 0.5 hr).

2.4 ICG loading in Fe₃O₄@mSiO₂-PEG

Briefly, 2 mg of ICG was pre-dispersed in 6 mL of anhydrous ethanol with strong ultrasonication. Thereafter, 60 mg of the as-prepared Fe₃O₄@mSiO₂-PEG nanoparticles were instantly poured to the prepared ICG-ethanol mixture. The ICG/ Fe₃O₄@mSiO₂-PEG solution was continuously and intensely stirred at a 37 °C oil-bath for 48 hr. At present, NIR II contrast agent, ICG was successfully adsorbed by the mesopores in the nanoshells. The as-prepared ICG encapsulated Fe₃O₄@mSiO₂-PEG nanocomposite (Fe₃O₄@mSiO₂- ICG/PEG) was render 3-times of centrifugal washing (10 min, 5000 rpm) with deionized water.

2.5 cMBP modification on Fe₃O₄@mSiO₂-ICG

At last, Fe₃O₄@mSiO₂-ICG/cMBP was constructed using condensation reaction. (Xie, et al., 2023). Firstly, 2 mg of cMBP protein was added into 2-(N-morpholino) ethane-sulfonic acid buffer (5 mL, 0.1 mol/L, pH=5.5). Secondly, 20 mg, 25 mg of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were simultaneously introduced into the above cMBP based mixture. An intense and continuous stirring was performed on this mixture for 2 hr at room temperature. Thereafter, 60 mg of Fe₃O₄@mSiO₂-ICG was added in the above cMBP based mixture with gent stirring for 12 hr. cMBP-decorated Fe₃O₄@mSiO₂-ICG was obtained by centrifugal washing (10 min, 5000 rpm) with H₂O and the finally Fe₃O₄@mSiO₂-ICG/cMBP nanoprobes were re-dispersed in H₂O and stored at 4℃.

2.6 Development of lymphatic metastasis model

All animal studies in this work were performed according to the Care and Use Guidelines for Lab Animals of Shanghai University and the procedures were also approved by the Animal-Ethics-Committee (Shanghai University). 6 weeks old, female Balb/c nude mice were bought from SLAC Lab Animal Co., Ltd. (Shanghai, China) and they were fed adaptively for 7 days before TNBC tumor bearing. In this work, a lymphatic metastasis-bearing mouse model was successfully established by injecting 25 μL, 5 × 10⁵ (PBS 1X) of MDA-MB 231 cells into the left-foot pad of naked mice. The metastatic model was monitored by touching and stroking the enlarged lymph nodes at each 3 days and histological-analysis on postinjection of 10 days. Successful lymphatic metastasis model can be acquired at 14 days post injection.

2.7 NIR II fluorescence bio-imaging of lymphatic metastasis

Above prepared lymphatic metastasis bearing-nude mice were firstly divided into 2 groups (n = 5). Real-time NIR II luminescence imaging procedure started at 15 min, 1 hr, 3 hr, 12 hr, 24 hr and 48 hr post tail-vein administration of 15 mg/kg Fe₃O₄@mSiO₂-ICG and Fe₃O₄@mSiO₂-ICG/cMBP, respectively. NIR II fluorescent bioimages were obtained via 1000-nm long-pass filter, 300-ms exposure time and 808-nm light excitation (400 mW/cm²).

2.8 MRI of lymphatic metastasis

The established lymphatic metastasis bearing-nude mice were firstly divided into 2 groups (n = 5). Real-time MRI procedure started at 15 min, 1 hr, 2 hr, 3 hr, 4 hr, 6 hr, 12 hr and 24 hr and 48 hr post tail-vein administration of 15 mg/kg Fe₃O₄@mSiO₂-ICG/cMBP, respectively. Mice with tail vein injection of Gd-DTPA for 15 min, 1 hr, 2 hr, 3 hr were conducted as the control group. T1-weighted bioimages were acquired using a 3.0 T magnetic resonance (MR) scanner.

3.1 Synthesis of Fe₃O₄@mSiO₂-ICG/cMBP

Fe₃O₄@mSiO₂-ICG/cMBP with superparamagnetic features were acted as the versatile carrier for accurate diagnosis of TNBC. The detailed step-by-step procedure for the preparation of this novel nanoprobe was illustrated in Fig. 1. Firstly, dispersible and hydrophilic Fe₃O₄ nanocores were fabricated by a conventional solvothermal strategy by using ethylene glycol as the redundant and solvent. FeCl₃·6H₂O was acted as the Fe source in the presence of sodium acetate. After 8 hr reaction under 200℃, the black Fe₃O₄ nanocrystals can be obtained. According to the images from transmission electron microscope (TEM) and scanning electron microscope (SEM), Fe₃O₄ nanoparticles with uniform diameter (∼70 nm) can be clearly observed (Fig. 2A, D). Secondly, the as-prepared Fe₃O₄ nanocrystals were coated with mesoporous silica nanoshell, in which CTAB served as the stabilizing surfactant on Fe₃O₄ surface and soft template for the silica nanocomponent formation in the sol–gel reaction. The CTAB template in the silica nanoshell was removed by an ion-exchange of ammonium nitrate, clearly, the mesoporous shell became and accordingly, mesoporous silica coated magnetite with core–shell nanostructure (Fe₃O₄@mSiO₂) with size of ∼ 200 nm could be successfully prepared (Fig. 2B, E). Meanwhile, from the dynamic laser scattering (DLS) data, the size increased from 71 nm to 195 nm after silica coating which were consisted with TEM and SEM photographs (Fig. 2G, H). Furthermore, The X-ray diffraction pattern of Fe₃O₄@mSiO₂ nanocomposites presented the characteristic peaks of Fe₃O₄ nanocrystals (JCPDS 9–0866), demonstrating that the composites of this Fe-based nanocrystals in core–shell nanoparticles (Fig. 2I). Besides, the mesopores observing under SEM (Fig. 2E), the porous structure of the prepared Fe₃O₄@mSiO₂ was apprized by a nitrogen-absorption/desorption isotherms curve. As displayed in Fig. 2J, according to the Brunauer-Emmett-Teller (BET), the specific surface area of Fe₃O₄@mSiO₂ was determined as ∼ 254 m²/g. The pore distributions of silica shell showed a clear unequal mesopore diameter of 8.8 nm (Fig. 2K). These data demonstrated that the core–shell nanocomposite can be further used for drug delivery nanoplatform (Rajarathinam, et al., 2024; Rajarathinam, et al., 2021; Jayaraman, et al., 2023). It has been extensively demonstrated that ICG possessed high shortwave infrared fluorescece imaging quality in the off-peak emmison tail (beyond 1000 nm) (Carr, et al., 2018; Hu, et al., 2020), in this work, we firstly evaluated the NIR II imaging feature of ICG solution with a 1000 nm long-pass filter (LP1000). Clearly, the increase in NIR II signalwas concentration dependent (Fig. S1). Thirdly, ICG dyes were absorbed in the mesoporous shell (Fe₃O₄@mSiO₂-ICG), and then PEG₂₀₀₀-NH₂ was grafted on the surface for the subsequent cMBP conjugation (Fe₃O₄@mSiO₂-ICG)/PEG). Finally, the targeting motifs were anchored on the out-layer of silica by a common condensation reaction (Fe₃O₄@mSiO₂-ICG/cMBP). The core–shell nanoprobes still maintained the core–shell morphology and mesoporous structure after ICG loading and cMBP grafting (Fig. 2C, F). Owing to the negative charges of ICG and PEG₂₀₀₀-NH₂, after ICG encapsulation and PEGylation, zeta potential increased. While after cMBP modification, zeta potential decreased which could be ascribed to the consumption of –NH₂ groups (Fig. 2L). Negative surface charge can reduce nanobio interactions, facilitating anionic nanoprobes to migrate from micro-vessels within the extracellular space and extensively distribute throughout the tumor tissue (Souri, et al., 2024). Besides, Fe₃O₄@mSiO₂-ICG/cMBP is then studied by Fourier-transform infrared spectrometer (FTIR), distinctly, a strengthened peak locating at 3300 cm ^-1 must be assigned to the new generate amide bond and two emerged peaks at 1229 cm ^-1,1090 cm ^-1 were originated from amino acids in cMBP (Fig. S2). Slight size variation can be visualized in Fe₃O₄@mSiO₂-ICG/cMBP and its diameter finally increases from 195 nm to ∼ 220 nm (Fig. S3), profoundly indicating the outlayer PEGylating and cMBP grafting. The results primarily indicated the successful development of Fe₃O₄@mSiO₂-ICG)/cMBP.

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Fig. 2

TEM images of Fe₃O₄ (A), Fe₃O₄@mSiO₂ (B) and Fe₃O₄@mSiO₂-ICG/cMBP (C). SEM images of Fe₃O₄ (D), Fe₃O₄@mSiO₂ (E) and Fe₃O₄@mSiO₂-ICG/cMBP (F).

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DLS results of Fe₃O₄ (G) and Fe₃O₄@mSiO₂ (H). XRD patterns of Fe₃O₄ and Fe₃O₄@mSiO₂ (I). The N₂ absorption/desorption plot of Fe₃O₄@mSiO₂ (J). Pore size distribution of Fe₃O₄@mSiO₂ (K). Zeta potential data of Fe₃O₄@mSiO₂ based nanoplatforms (L).

3.2 Characterization of Fe₃O₄@mSiO₂-ICG/cMBP

Recently, mesoporous silica nanoparticles (mSiO₂) are increasingly put forward as promising candidate for drug delivery nanocarriers and thus prominently improving the saturation solubility of poorly dissolve nanocompounds (Ibrahim, et al., 2020). We then fabricated the mSiO₂ nanocarriers with the same size of Fe-based core–shell nanoplatform (Fig. S4, S5). In the meantime, these silica nanospheres also had similar specific area and pore size (Fig. S6). Compared with ICG loaded and cMBP anchored mSiO₂ (mSiO₂-ICG/cMBP) that dispersed in pure water to a variety of dosage, no apparent sediments or flocs were found in Fe₃O₄@mSiO₂ and Fe₃O₄@mSiO₂-ICG/cMBP groups, even if the concentration reaches up to 1500 μg/mL (Fig. 3A-C). These results demonstrated that our nanoprobes of Fe₃O₄ nanocrystals with porous outer shells, ICG loading and targeting motif binding had an excellent aqueous dispersion behavior. Understanding the nanoparticles size variations in various physiological media plays a vital role in determining the following bio-application fate, we then investigated the size stability of our nanoprobes toward different biological buffers. As depicted in Fig. 3D-E, obviously, no evident size fluctuations of mSiO₂-ICG/cMBP, Fe₃O₄@mSiO₂ and Fe₃O₄@mSiO₂-ICG/cMBP were monitored even after 48 h immersion in 0.9 % NaCl, PBS, and cell medium, respectively, confirming the stable size feature of this nanoprobes. Furthermore, the hemolytic properties of our synthetic Fe-based nanoparticles were conducted. The hemolysis rate of Fe₃O₄@mSiO₂-ICG/cMBP slightly increased 3.8 % to 12 % when the concentration of these nanoprobes were set from 500 μg/mL to 1500 μg/mL, and no significant discrepancy were discovered among the three nanoplatforms (Fig. 3G, S7), laying the solid foundation for subsequent in vivo research. Additionally, the c-Met binding affinity was subsequently studied, FITC labelled Fe₃O₄@mSiO₂ with cMBP modification (Fe₃O₄@mSiO₂-FITC/cMBP) was prepared in advance, then it was immersed in c-Met enveloped microtiter plate in a dose-dependent way. The absorbance data showed that cMBP modified nanoprobes gradually increased with the incubation time. According to the reported works, cMBP could specifically bind with c-Met against hepatocyte growth factor (HGF) (Wu et al., 2021). When Fe₃O₄@mSiO₂-FITC/cMBP and HGF was introduced to the c-Met coated microplate, our nanoprobes inhibited the affinity of c-Met to HGF (Fig. 3H). Altogether, cMBP might contribute to strong binding affinity between nanoprobes and overexpressed c-Met in TNBC cells.

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Fig. 3

Representative digital photos of mSiO₂-ICG/cMBP (A), Fe₃O₄@mSiO₂ (B), and Fe₃O₄@mSiO₂-ICG/cMBP (C). Size variations of mSiO₂-ICG/cMBP (D), Fe₃O₄@mSiO₂ (E), and Fe₃O₄@mSiO₂-ICG/cMBP (F) dispersed in 0.9 % NaCl, PBS, and cell medium. (G) Hemolysis rate of mSiO₂-ICG/cMBP, Fe₃O₄@mSiO₂, and Fe₃O₄@mSiO₂-ICG/cMBP. (H) Binding activity of Fe₃O₄@mSiO₂-FITC/cMBP or Fe₃O₄@mSiO₂-FITC/cMBP+HGF to c-Met at various concentrations.

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3.3 Cell viability and cellular internalization in vitro

It has been confirmed that metastatic squamous cell carcinoma (SCC) cells overexpress c-Met protein (Ozawa, et al., 2015). In the constructed murine SCC model, the high levels of angiogenesis factors and on tumorigenesis and metastasis enhancement were is accompanied by with HGF/c-Met pathway (Raj, et al., 2022) Based on this, cell viabilities and cellular internalization of cMBP-conjugated nanoprobes at different doses were studied against MDA-MB 231 (TNBC cell line) and SCC7 cells. As shown in Fig. 4A,B,D,E, even the incubated concentration set as high as 800 μg/mL and the co-culture time extended as long as 24 hr, the cell viabilities of both two kinds of tumor cells still remained over 90 %, pronouncedly confirming that the Fe₃O₄@mSiO₂-cMBP nanocomposites possessed perfect biocompatibility. In addition, negligible cell toxicity was discovered in normal macrophage (Raw 264.7) when the concentration of Fe₃O₄@mSiO₂-ICG/cMBP increased to 600 μg/mL (Fig. S8), pronouncedly reflecting the exceptional biocompatibility in vitro. Further, owing to c-Met receptor is known to be overexpressed in these two carcinoma cells, thus, cMBP-conjugated Fe₃O₄@mSiO₂ (labeled with FITC) was expected to be effectively targeted the cancerous cells. Fig. 4G, C shows the CLSM images and quantitative fluorescence intensities of MDA-MB 231 cells after incubated with Fe₃O₄@mSiO₂ and Fe₃O₄@mSiO₂-cMBP for 6 hr, respectively. These two nanocomposites appeared as green light and the cMBP anchored group had much stronger luminescence intensity than the exposed surface of Fe₃O₄@mSiO₂ nanocomposites, indicating that c-Met receptor-mediated tumor cell recognition accelerated the cellular internalization of Fe₃O₄@mSiO₂-cMBP. Whereas, in the MDA-MB 231 cells pre-treated with cMBP groups for blocking c-Met, the green fluorescence had decreased twelve folds (Fig. 4C). We also conducted cellular endocytosis of ICG loaded nanoprobes (Ex 633 nm, Em, 750 nm) toward MDA-MB 231 by CLSM, as shown in Fig. S9, in comparison with other two groups, prominent high red fluorescence from ICG can be detected in Fe₃O₄@mSiO₂-ICG/cMBP. In addition, similar fluorescent trend was found in SCC7 groups, while in sharp contrast, Fe₃O₄@mSiO₂-cMBP treated TNBC cell showed relative two times cellular endocytosis than that of SCC7 cell (Fig. S10). The underlying mechanism of stronger green fluorescence in 4 T1 cells might be atributed to relatively higher c-Met expression in 4 T1 cells (Fig. S11). All above findings demonstrated the more effective diagnostic ability of cMBP-conjugated nanoagents toward TNBC tumors.

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Fig. 4

Cell viability of MDA-MB 231cells after treated with mSiO₂-cMBP, Fe₃O₄-cMBP and Fe₃O₄@mSiO₂-cMBP at different concentrations (A) or various incubation periods (B). Cell viability of SCC7 cells after treated with mSiO₂-cMBP, Fe₃O₄-cMBP and Fe₃O₄@mSiO₂-cMBP at different concentrations (D) or various incubation periods (E). Quantitative fluorescence intensity (C) and CLSM images (G) of Fe₃O₄@mSiO₂, Fe₃O₄@mSiO₂-cMBP and Fe₃O₄@mSiO₂-cMBP+cMBP treated MDA-MB 231 cells. Quantitative fluorescence intensity (F) and CLSM images (H) of Fe₃O₄@mSiO₂, Fe₃O₄@mSiO₂-cMBP and Fe₃O₄@mSiO₂-cMBP+cMBP treated SCC7 cells.

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3.4 NIR II fluorescence imaging and MRI properties in vitro and in vivo

In vitro NIR II luminescent bioimaging capacity was evaluated by a home-made NIR II fluorescence imaging system for small animal. In accordance with ICG, the strong off-peak emmison (beyond 1000 nm) of Fe₃O₄@mSiO₂-ICG/cMBP was apprised (Fig. S12). Expectedly, with the increase of dosage, the NIR II luminescence intensity of of Fe₃O₄@mSiO₂-ICG/cMBP, were increasingly brighter under 808-nm continuous laser illumination (P=0.5 W/cm², 100 ms, 1000 nm long-pass filter) (Fig. 5A). A highly linear correlation with (R² = 0.999) of mean fluorescence intensity at a range of dose from 0.345 to 4.189 μg/mL (Fig. 5B). Besides, T1-weighted MRI pictures of the fabricated Fe₃O₄@mSiO₂-ICG/cMBP nanocomposite were obtained by using a MR scanner (3.0 T). As displayed in Fig. 5C, the novel Fe₃O₄ based nanocomposite presented an apparent Fe concentration-dependent brightening effect, profoundly demonstrating the sensitive T1 weighted contrast effects. Additionally, Fig. 5D clearly illustrates the excellent linear relationship (R² = 0.999). The in vitro NIR II/MRI results suggested that the Fe₃O₄@mSiO₂-ICG/cMBP nanocomposite had the huge potential to be applied as a contrast agent for sensitive fluorescence imaging and MRI in malignant tumor discrimination.

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Fig. 5

(A) NIR II fluorescent and (C) MRI bioimages of the fabricated Fe₃O₄@mSiO₂-ICG/cMBP at various concentrations. Plots of the mean fluorescence intensity (B) and 1/T1(S^-1) (D) vs. the dosage of Fe₃O₄@mSiO₂-ICG/cMBP.

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Aforementioned excellent fluorescence feature of Fe₃O₄@mSiO₂-ICG/cMBP inspired us to evaluate the corresponding in vivo NIR II fluorescence imaging effect on the TBNC lymphatic metastasis bearing nude mice. To better address the precise recognition of our nanoprobe (with cMBP) toward TNBC tumor tissues, we selected Fe₃O₄@mSiO₂-ICG (without cMBP) as the control agent. After caudal-vein administration of these two nanocomposites for 15 min, bioimaging was executed as depicted in Fig. 6A. Apparently, NIR II fluorescent signals in the lymphatic TNBC metastasis could be gradually observed in both “with” and “without” groups at 1 h post-administration. Evidently, mice in the latter group only presented feeble signal, expectedly, stronger fluorescence was visualized in cMBP conjugated group, especially at 3 h, when the total contour of the small lesion can be successfully distinguished. Then, in the TNBC metastasis tissues of the two groups, NIR II light decayed from this timepoint. Concurrently, Fe₃O₄@mSiO₂-ICG/cMBP produced relatively higher signal to background ratios (SBR) when compared to Fe₃O₄@mSiO₂-ICG at every time period, particularly, an optimal SBR value was obtained in the “with” group at 3 h (13.6 ± 1.5 vs. 5.9 ± 1.4) (Fig. 6C). This result was substantially due to more efficacious tumor concentrated effect of Fe₃O₄@mSiO₂-ICG/cMBP. Futher, after tail-vein injection of Fe₃O₄@mSiO₂-ICG/cMBP (15 mg/kg), the vital organs were imaged under NIR II fluorescence imaging system, even massive nanoprobes accumulated in the reticuloendothelial system at 12 hr, they were approximately metabolized at 48 hr (Fig. S13), primarily reflecting the low side effect in vivo. To further explore the feasibility of our nanoprobes for MRI, gadopentetic acid (Gd-DTPA), as a clinical MRI contrast agent, was used as the control. After intravenous administration of above two agents, as illustrated in Fig. 6B, metastasis area became strongest in Gd-DTPA treated mice at 2 h post-administration, while almost all T1 weighted signal drastically vanished at 3 h, this performance might be assigned to the low blood retention time of this complex. Exhilaratingly, the MRI signal of the targeted group appeared at 15 min post-administration and achieved at the maximum level at 3 h, then it slowly started to decrease along with time extension. The highest tumor enrichment time point was coincident with fluorescence imaging. In the meantime, compared with Gd-DTPA, Fe₃O₄@mSiO₂-ICG/cMBP still maintained a significantly higher intensity even after 6 h post-administration. Even the relative signal intensity of targeted group was slightly less than Gd-DTPA group (12.4 vs. 14.7) at 2 h post injection, inspiringly, it intensely increased to three times higher than that of Gd-DTPA (Fig. 6D). This feature can be attributed to PEG coatings on Fe₃O₄@mSiO₂-ICG/cMBP. It shielded nanoprobes from aggregation, phagocytosis as well as prolonging systemic blood circulation time (Suk, et al., 2016). All above data illustrated that the robust, specific binding affinity of cMBP-grafted nanoprobe toward c-Met overexpressed TNBC carcinoma and it can be exploited as an effective contrast nanoagent for MRI/NIR II fluorescence imaging.

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Fig. 6

(A) In vivo NIR II fluorescent bioimages of lymphatic metastasis bearing naked mouse after caudal-vein injection of Fe₃O₄@mSiO₂-ICG (without) or Fe₃O₄@mSiO₂-ICG/cMBP (with) at different time periods. (B) MRI bioimages of lymphatic metastasis bearing naked mouse after intravenous administration of Gd-DTPA and Fe₃O₄@mSiO₂-ICG/cMBP for different times. (C) SBR values of Fe₃O₄@mSiO₂-ICG (without) or Fe₃O₄@mSiO₂-ICG/cMBP (with) at different time periods. (D) Relative MRI signal intensities of Gd-DTPA and Fe₃O₄@mSiO₂-ICG/cMBP at 15 min, 1 hr, 2 hr and 3 hr.

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3.5 Biocompatibility assessment in vivo

Additionally, in vivo biocompatibility of Fe₃O₄@mSiO₂-ICG/cMBP was determined. After intravenous administrated our nanoprobe for 7 days and 30 days, blood samples and major organ tissue were collected. Blood bio-chemical tests and H&E staining studies were conducted for exploring the biosafety of Fe₃O₄@mSiO₂-ICG/cMBP in vivo. Here, the serum bio-chemistry factors include alanine transaminase (ALT, Fig. 7A), alkaline phosphatase (ALP, Fig. 7B), aspartate transaminase (AST, Fig. 7C), total bilirubin (TBIL, Fig. 7D), serum creatinine (CRE, Fig. 7E), blood urea nitrogen (BUN, Fig. 7F) and creatine kinase (CK, Fig. 7G). Collectively, in comparison with control group (PBS treatment), there were insignificant discrepancy in the seven indexes of the Fe₃O₄@mSiO₂ based groups at 7 days and 30 days post-administration. In the bargain, both scale-down and full-size H&E-stained-images of the main normal organ tissues (including heart, liver, spleen, lung and kidney) revealed no apparent pathological injury or normal cellular damage were discovered (Fig. 7H, S14), preliminarily illustrating the excellent in vivo histocompatibility of Fe₃O₄@mSiO₂-ICG/cMBP as well as its potent bio-application for TNBC patients.

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Fig. 7

Serum bio-chemistry studies, including factors like ALT (A), ALP (B), AST (C), TBIL (D), CRE (E), BUN (F), CK (G)of healthy mice after 7 days or 30 days post-administration of Fe₃O₄@mSiO₂-ICG/cMBP. (C) H&E-stained-images of the main organs tissues (including heart, liver, spleen, lungs, kidney) resected from mice after 7 days, 30 days treatment of Fe₃O₄@mSiO₂-ICG/cMBP. PBS treated mice were set as control.

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