Preparation and characterization of HM/D-I-BL
The procedure for the synthesis of HM/D-I-BL is illustrated in Fig. S1. First, HM-SiO2 was obtained via the reduction of KMnO4 with organosilia existing on freshly prepared silica NPs. Then, Na2CO3 solution was incubated with HM-SiO2 NPs to dissolve silica for achieving hollow mesoporous MnO2 (HM).36 Transmission electron microscope (TEM) images of HM clearly revealed the spherical morphology and the hollow structure of HM (Fig. S1). The hollow structure of HM was further confirmed by high-angle annular dark-field scanning TEM (HHAADF-STEM)-based elemental mapping (Fig. S1). Fig. S2 showed Mn 2p and O 1s XPS spectrum of HM. XRD results demonstrated that HM is amorphous (Fig. S3). Next, the HM were mixed with certain concentration of DOX to form H-M/D. To achieve the effective encapsulation of ICG in the lipid bilayer of liposomes, a hydrophobic modification was first performed by conjugating the amino groups on octadecylamine (ODA) with the sulfonic groups on ICG. Then the extractive membrane proteins and the ICG-loaded liposomes were mixed by successive extrusion to form a stable biomimetic liposome nanomedicine (I-BL). In the process of surface functionalization, the I-BL were mixed with H-M/D by coextrusion method to produce biomimetic cell membrane-coated NPs (HM/D-I-BL). To characterize the specific nanostructure of HM/D-I-BL, TEM imaging was performed. The TEM imaging of HM (Fig. 1A) and HM/D-I-BL (Fig. 1 B) demonstrated that the prepared biomimetic NPs are core/membrane nanostructures, when compared to HM, a bright film was formed outside the core. Dynamic light scattering (DLS) analysis showed that the mean size of HM and HM/D-I-BL were 121.67+0.98nm and 174.64±1.37 nm, respectively (Fig. 1C). The zeta potentials of HM, I-BL, H-M/D, and HM/D-I-BL were ‒21.3±1.5 mV, ‒27.1±1.6 mV and ‒24.8±1.0 mV, respectively (Fig. 1D), indicating HM/D-I-BL is generally spherical in shape with good mono-dispersity and suitable for its potential application in vivo. The surface area and average pore diameter of HM were determined to be 121.529m2/g and 3.932 nm, respectively, by Brunauer–Emmett–Teller (BET) measurement (Fig. 1E). The formation of HM/D-I-BL and H-M/D, as well as I-BL were investigated by measuring their UV–visible absorption. Fig. 1F showed that HM/D-I-BL has an absorption band peak around 780 nm indicating that ICG was successfully loaded. In comparison, I-BL, H-M/D, and HM/D-I-BL showed a UV–visible absorption band around 500 nm, which could be attributed to DOX. Next, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to verify whether the extracted 4T1 tumor membrane proteins were efficiently embedded in the phospholipid bilayer on the surface of NPs. As shown in Fig. 1G, the protein profile of HM/D-I-BL was consistent with that of the 4T1 cell membrane, indicating that 4T1 cell membrane proteins were successfully decorated on the surface of HM/D-I-BL. Moreover, the DOX encapsulation efficiency (EE) of HM/D-I-BL increased from 25.15% to 64.79% when the weight ratio of DOX:HM increased from 0.5 to 4 (Fig. 1H). Further, the DOX release behavior under laser irradiation was investigated when NPs were exposed to GSH solution with different PH. It was found that HM/D-I-BL+GSH showed little DOX release in the absence of laser irradiation. However, HM/D-I-BL+GSH exhibited a remarkably enhanced DOX release after laser irradiation, which was closely related to the intensity and time of irradiation (Fig. 1I, S4A, S4B). Besides, the amount of DOX release in different PH solutions showed no significant difference indicating the effect of reductive environment on the drug release profile is important than pH values. In general, the drug release mechanism might be the disorder and fusion of lipid bilayer caused by high temperature after laser irradiation.
Targeting capability characterization
It has been proven that NPs modified with cancer cell membrane could inherit homologous targeting and immune-evading characteristics from their source cancer cells.37,38 To investigate the specific homotypic targeting ability of HM/D-I-BL to homologous cancer cells, the uptake of DOX and ICG of HM/D-I-BL in 4T1 cells was evaluated by confocal laser scanning microscope (CLSM) imaging and flow cytometry. After incubation at 37℃ for 2 h with HM/D-I-L and HM/D-I-BL, a higher amount of HM/D-I-BL penetrated the 4T1 cells, which exhibited a stronger fluorescence signal when compared to the HM/D-I-L group (Fig. 2A, 2B), indicating that HM/D-I-BL had homotypic targeting ability. In order to further verify the specific cellular uptake, HM/D-I-BL was evaluated by incubating the NPs with several different cell lines, including 4T1, bEnd.3, HeLa, MCF-7 and MDA-MB-468 cells. The results revealed that the fluorescence intensity of DOX in the 4T1 cell group was higher compared to that of other cell lines (Fig. 2C). The higher uptake efficiency of 4T1 cells confirmed the excellent active targeting ability of HM/D-I-BL, which benefit from cellular adhesion molecules including EpCAM, N-cadherin and galectin-3 on cancer cell membranes mediated specific adhesion junctions between homologous cells.16,17 In addition, HM/D-I-L and HM/D-I-BL uptake by RAW264.7 cells were further investigated by CLSM imaging and flow cytometry. As shown in Fig. 2D, only a very small amount of HM/D-I-BL was internalized by RAW264.7 macrophages. Quantitative analysis by flow cytometry showed 0.51-fold fluorescence as compared to that for HM/D-I-L treated cells (Fig. 2E), suggesting the good immune escape ability of HM/D-I-BL. The excellent homologous cellular uptake and immune escape ability of HM/D-I-BL may make it a promising delivery system for TNBC precision nanomedicine.39
O2 and ROS generation evaluation in vitro
Next, the TME-responsive capability of HM/D-I-BL as a mutifunctional nanoplatform at the in vitro level was investigated (Fig 3A). The capability of generating O2 in vitro by HM and HM/D-I-BL upon reaction with H2O2 (10 mM) and GSH (10 mM) at endogenous levels was first evaluated. Fig.3B showed that HM could rapidly trigger O2 generation by a HM concentration-dependent manner. The O2 generation in the presence of HM/D-I-BL was measured to clarify whether laser irradiation would promote the process. As shown in Fig. 3B-D, the O2 generation triggered by HM/D-I-BL with laser irradiation was the same with HM, but no obvious O2 generation was observed form HM/D-I-BL without laser irradiation. O2 bubbles produced by HM/D-I-BL were visible by eye observation after different treatments (Fig. S5). These results suggested that biomimetic membrane modification can block the reaction between the MnO2 core and external environment, reducing the production of O2. Whereas, laser irradiation inducing the outer membrane phase transition took place at a certain temperature, which resulted in an increase on O2 production.
In addition, 1O2 generation from H2O2 solution was tested in the absence or presence of laser irradiation (808 nm laser, 1 W/cm2, 5 min). The results showed the absorbance of solution in the presence of HM/D-I-BL+DPBF without significant change, while obvious decrease was observed upon laser irradiation, indicating 1O2 production ability was remarkably enhanced due to more O2 production from H2O2 and that laser irradiation is an essential element in the process of 1O2 production (Fig. 3E). Next, the intracellular ROS generation were observed by using 2’,7’-dichlorofluorescin diacetate (DCFDA) probe40. There is no obvious fluorescence in control group just exposed to DCFDA, as well as DOX and HM group with laser irradiation showed weak fluorescence. Among the other three groups, the intensity of fluorescence in ICG, HM/D-I-L and HM/D-I-BL suffered from laser irradiation was increased in turn, which were highly consistent with the targeting capability and O2 generation, further verifying the advantages of HM/D-I-BL.
Finally, after exposed to GSH MnO2 could be reduced to Mn2+ with excellent Fenton-like activity to generate highly toxic hydroxyl radical (·OH) from H2O2 in the presence of HCO3− for chemodynamic cancer therapy. To investigate the Fenton-like reaction, we utilized the methylene blue (MB) as an ·OH generation probe. Compared with MB solution, the color and characteristic absorbance of MB+Mn2+ solution was almost vanished, revealing the Fenton-like reaction (Fig. S6). As shown in Fig 3G, the HM/D-I-BL without laser pretreatment and GSH failed to induce Fenton reaction, while an obvious Fenton-like MB degradation was observed in the presence of laser pretreatment and GSH. These results indicated that HM/D-I-BL only with laser pretreatment could release Mn2+ in response to GSH, which subsequently generates ·OH via Fentonlike reaction. In general, the O2 and ROS production, as well as ·OH generation mechanism might be because the HM core was exposed to the outer environment caused by phase transition of lipid bilayer, associated with an increase of the outer membrane permeability after ICG laser treatment induced hyperthermia.41
Enhanced chemotherapy and phototherapy efficacy in vitro
Increasing evidence showed that hypoxic TME is responsible for the limited chemotherapy and PDT efficacy for treatment of solid tumors. Considering that HM/D-I-BL can successfully catalyze H2O2 into O2, the cytotoxicity of HM/D-I-BL on 4T1 cells was investigated. As shown in Fig. 3H, low dose DOX and HM/D-I-BL showed no obvious cytotoxicity to 4T1 cells for 24 h, whereas HM/D-I-BL plus laser irradiation significantly induced cell death, especially at increased concentration. These results confirmed that 4T1 cells were resistant to low dose DOX, while O2 and ROS generation after laser irradiation could synergize with DOX and eliminate hypoxia-induced DOX resistance, which is consistent with previous studies.42-44 The in vitro phototherapy efficiency of the NPs was further tested by cell viability assay. The cell viability in ICG (20 μg/mL) group was 103.25±8.58%, whereas in the ICG+Laser and HM/D-I-BL+Laser group, the viability was 56.39±6.37% and 20.63±4.04%, respectively (Fig. 3I), indicating that HM/D-I-BL plus laser irradiation exhibited excellent phototherapeutic effects on 4T1 cells. These in vitro results implied potential advantages for in vivo applications.
MnO2 is known to be stable under neutral and basic pH, but can be decomposed into Mn2+ which has been widely utilizing as a contrast agent for MRI under reductive enviroment.45-47 Interestingly, the obtained T1-weighted signals of HM/D-I-BL demonstrated a significant concentration-dependent contrast enhancement effect (Fig. 4A). The T1-weighted signal of HM/D-I-BL NPs has not significant difference between pH 6.0 and 7.4 under reductive environment (GSH concentration: 10 mM), and the longitudinal relaxivity (r1) value was calculated to be 2.344 and 2.114 mM-1s-1, respectively (Fig. 4B, S7). The remarkably increased relaxivity of the NPs in GSH was attributed to the decomposition of MnO2 into paramagnetic Mn2+ indicating the switch of T1-weighted MR imaging from “OFF” to “ON” states. The T1-weighted MR images of TNBC were examined after intravenous injection of HM/D-I-L or HM/D-I-BL into mice with TNBC at 0, 2, 4, 8, 12, and 24 h (Fig. 4C). TNBC tissue and normal tissue showed no difference in T1-weighted signal intensity before injection. However, the intensity was significantly increased in the TNBC area from 4 h (RSI=1.46±0.18) to 8 h (RSI=2.34±0.31) after injection with HM/D-I-BL but not with HM/D-I-L (Fig. 4D). Moreover, the enhanced T1-weighted signal intensity in TNBC areas lasted for 24 h after injection with HM/D-I-BL (RSI=1.81±0.24). These results further confirmed the accumulation of HM/D-I-BL in 4T1 tumor tissue and responsive imaging in TEM, which may have great potential for MRI diagnosis of TNBC.
After the establishment of the TNBC model in Balb/c mice, the distribution of HM/D-I-L and HM/D-I-BL in mice was evaluated by NIR imaging. The fluorescence of ICG distributed widely throughout the mouse body after 2 h injection of the NPs (Fig.4E, 4F). Subsequently, HM/D-I-BL showed remarkable accumulation in the tumor area in mice with TNBC from 2 to 12 h. In contrast, the mice with HM/D-I-L did not show obvious accumulation in the tumor area, which indicated excellent in vivo tumor targeting of the NPs. To further prove the in vivo uptake of the NPs, ex vivo imaging of excised major organs and tumors at 24 h post-injection were investigated (Figure 4G, 4H), and the results were consistent with the previous fluorescence imaging results.
Photothermal properties evaluation in vivo
Another important property of HM/D-I-BL is its adjustable photothermal effect. Temperature changes in deionized water, ICG, and HM/D-I-BL aqueous solutions under 808 nm laser irradiation at 1.0 W/cm2 were initially investigated (Fig. S8). The initial temperature of deionized water, ICG, and HM/D-I-BL solution was 27.9°C, 28.6°C, and 27.7°C, respectively. The solution temperature increased precisely by 22.0°C and 18.8°C (ICG:20 μg/mL). On the contrary, the temperature of pure water exhibited no obvious change. Given the ideal in vitro results, the photothermal effect of NPs were further examined in vivo. TNBC tumor-bearing mice were randomly divided into three groups and treated with saline+Laser, HM/D-I-L+Laser, and HM/D-I-BL+Laser, respectively. Among the three groups, the temperature of tumor site in HM/D-I-BL+Laser group reached 58.2°C, which was higher than those of other groups, indicating efficient conversion of light to heat by the HM/D-I-BL NPs to induce elimination of cancer cells (Fig.5A-B).
Therapeutic efficacy evaluation in vivo
Mice with TNBC were randomly divided into seven groups: control, I-BL, HM/D, HM/D-I-BL, I-BL+Laser, HM/D+Laser, and HM/D-I-BL+Laser. Tumor volumes of each group were ~100 mm3 before treatments. Tumors were irradiated with 808 nm laser for 5 min (1 W/cm2) after 2 h injection of the NPs. The therapeutic efficacy of HM/D-I-BL+Laser-enhanced chemotherapy and phototherapy was further evaluated by monitoring tumor weight and volume. On day 15, the tumors of TNBC bearing-mice treated with HM/D-I-BL+Laser showed significant regression (p<0.001) (Figure 5C). Besides, tumor regressions induced by HM/D and HM/D+L groups were remarkably weaker than that of HM/D-I-BL group, suggesting the active targeting ability of HM/D-I-BL. However, the tumor weight did not differ between HM/D-I-BL and I-BL plus laser irradiation groups, probably due to from the effective chemotherapy or phototherapy (Fig. 5D). The results of tumor volumes among seven groups were consistent with that of tumor weight (Fig. 5E). These findings due to the hypoxia alleviation of tumor tissue mediated by MnO2 plus chemotherapy and phototherapy is important for the remarkably improved therapeutic efficacy of HM/D-I-BL plus laser irradiation.
The high-quality pathological H&E tumor slices on day 15 after treatments are shown in Fig. 5G. The corresponding H&E and TUNEL sections showed larger tumor necrosis area and more cell apoptosis with HM/D-I-BL plus laser irradiation treatment, which suggested more complete tumor damage with HM/D-I-BL+Laser enhanced chemotherapy and phototherapy effect. These results suggest that HM/D-I-BL may be a powerful chemical and photo agent, superior to HM/D and I-BL plus laser irradiation, because HM/D-I-BL significantly improved tumor hypoxia resulting from MnO2-triggered H2O2 decomposition and increased the concentration of drugs in the tumor site induced by biomimetic modification.
To evaluate the potential toxicity of HM/D-I-BL treatment, the body weight loss of mice was used as an indicator for treatment-induced toxicity (Fig. 5F). The result showed the body weight of mice did not differ with various treatments. To further assess the in vivo safety of HM/D-I-BL, the main organs of mice on day 15 post-treatment were excised and analyzed. H&E-stained sections of the five tissues (heart, liver, spleen, lung, and kidney) showed no apparent lesion (no necrosis, edema, inflammatory infiltration or hyperplasia) with saline or the NPs injection (Fig. S9), suggesting chemotherapy and phototherapy for TNBC with HM/D-I-BL treatment did not induce significant damage in major organs. Therefore, the FI and MRI monitored chemotherapy and phototherapy synergistic therapy mediated by HM/D-I-BL with advanced tumor inhibition effect and good biocompatibility was promising in anti-TNBC treatments.