Preparation of polyacrylic acid modified cerium nanoparticles (CP)
5 mL solution containing 1 M Ce2(CO3)3 (with nitric acid treatment) were mixed with 10 mL polyacrylic acid (0.05 M). After ultrasonic treatment for 10 min, 30 mL of 30% ammonium solution were added and the mixture kept stirring for 24 h. The prepared solution was centrifuged at 4000 rpm for 30 min and washed with ultra-pure water to remove insoluble precipitation. The PAA-modified CeO2 NPs were obtained after centrifugation at 12,000 rpm for 30 min. After dispersing in the ultra-pure water and ultrasound treatment for 10 min, the solution was frozen to get powder.
Preparation of chlorin e6 and CS-1 loaded cerium nanoparticles (CPCC)
For loading Ce6, 1 mL CeO2 NPs solution (1 mg/mL) were mixed with 0.6 mg of EDC and 1.2 mg of NHS. After stirring for 30 min, 1 mL of Ce6 solution (0.05 mg/mL) was added to the mixture and kept stirring for 24 h. Then, the mixture was centrifugated at 12,000 rpm for 10 min to remove the excess Ce6, EDC, and NHS. The finally obtained precipitation was resuspended in the deionized water.
For the CS-1 load, 1 mg of CS-1 was dissolved in 1 mL of dimethylsulfoxide (DMSO). Then, 0.1 mL CS-1 solution was added dropwisely into 1 mL (1 mg/mL) CeO2 NPs solution or Ce6 loaded CeO2 NPs. After stirring for 24 h, the solution was centrifugated at 12,000 rpm for 10 min to remove the excess CS-1. The obtained precipitation was resuspended in the ultra-pure water.
Preparation of erythrocyte-cancer hybrid membrane (RBC-231)
RBC membranes were prepared following our previous report [23]. The whole blood collected from female BALB/c mice (6–8 weeks) was centrifuged at 3,500 rpm for 5 min at 4 °C. The precipitated RBCs were washed with cold 1× PBS for 3 times. Then, 0.25× PBS was added into cells and incubated on the ice for 30 min. The RBC membranes were obtained after centrifugation at 12,000 rpm for 5 min. After washing with cold 1× PBS for 3 times, the collected membranes were stored at – 80 °C.
MDA-MB-231 cell membranes were prepared following the instruction of the membrane protein extraction kit. In brief: Tumor cells washed with ice-cold PBS were suspended in the membrane protein extraction reagent A containing 1 mM PMSF for 15 min at 4 °C. After through 3 freeze–thaw cycles and 50 W sonication for 3 min, the mixture was centrifuged at 3000 rpm for 10 min at 4 °C. The supernatant was centrifuged at 13,000 rpm for 30 min at 4 °C to obtain cell membrane. After mixing the RBC membrane and MDA-MB-231 membrane with equal weight, the mixture was treated with ultrasonication for 10 min at 37 °C to complete membrane fusion.
Preparation of the hybrid membrane coated CPCC (CPCCM)
The obtained CPCC was ultrasonic for 30 min to redisperse into monodisperse system. Then, the mixture of CPCC and hybrid membrane with a weight ratio of 2:1 was add into an ultrasonic bath for 5 min to obtain hybrid membrane-coated CPCC NPs(CPCCM NPs). The uncoated membrane was removed through centrifugation at 12,000 rpm for 30 min at 4 °C. The CPCCM NPs were washed with PBS until no protein was detected in the supernatants.
Characterization of material and RBC-231 hybrid membrane
For characterization of material, the morphology was assessed using a high-resolution JEOL JEM-2100Plus TEM (Hitachi Scientific Instruments, Japan). The FT-IR spectra were recorded with a Thermo-Nicolet Nexus 6700 FT-IR spectrometer (USA). The UV–Vis spectra were measured with a DU800 spectrometer (Beckman Coulter Inc., USA). The size and zeta potential of nanomaterial were measured with a Zetasizer Nano analyzer (Malvern Nano Series, UK). The chemical states of the elements were measured with an X-ray photoelectron spectroscopy (XPS) (ULVAC-PHI Inc., Japan).
A fluorescence assay was illustrated to characterize the extent of membrane fusion. Briefly, the DiI labeled RBCM and DiO labeled 231 M were mixed with equal weight. The fluorescence of the hybrid membrane was imaged under confocal laser scanning microscope (CLSM).
SDS-PAGE was employed to characterize the integrity of membrane proteins. Briefly, RBCM, 231 M, HM, and CPCCM were lysed with membrane protein extraction reagent B containing 1 mM PMSF. The protein concentration of all samples was detected using the BCA Kit, followed by proteins (20 μg) denature at 95 °C for 10 min. The proteins were separated in the 12% SDS-PAGE gel and stained using Coomassie Brilliant Blue solution.
Detection of peroxidase-like activity and PDT of CPCCM NPs in vitro
1 mM TMB and 5 mM H2O2 were sequentially added into 10 μg/mL of CPCCM NPs solution. The absorbance values of samples at 652 nm (the characteristic peak of ox-TMB) were monitored at different time points. H2O2 of series concentration was used for kinetic study of peroxidase activity of CPCCM NPs.
The dissolved O2 concentration was detected using dissolved O2 meter. Briefly, 500 μg CPCCM NPs were added to 10 mL 1 mM H2O2 solution (pH6.8) and the dissolved O2 concentrations were measured at different time point. In parallel sample, the amount of H2O2 consumption was determined after adding ammonium molybdate (final concentration of 2.4 mM) by recording the absorbance values at 330 nm.
The generation of 1O2 by Ce6, CPCC, and CPCCM was detected using the trapping agent of TEMP. Briefly, 1 mL of Ce6, CPCCM, and CPCC NPs (with equivalent concentration of 5 μg/mL Ce6) was mixed with 100 μL of TEMP (640 μM). After with 660 nm laser irradiation (0.2 W/cm2, the 1O2 signal was immediately detected by the ESR. In addition, the quantitative detection of 1O2 was detected using SOSG probe. Ce6, CPCC, and CPCCM NPs (equivalent concentration of 5 μg/mL Ce6) were mixed with SOSG (5 μM) dissolved in PBS (pH 7.4) following with 660 nm laser irradiation (0.2 W/cm2). The fluorescence intensities of samples at 530 nm were recorded at predetermined time intervals (Ex = 488 nm).
Loading and releasing behavior in vitro
To determine loading capacity (LC) and encapsulation efficiency (EE) of CS-1 and Ce6, different amounts of CS-1 and Ce6 were redispersed in CP NPs solution. The loading method is consistent with the above CPCC NPs synthesis protocol. Final nanomaterials were centrifuged at 12,000 rpm for 10 min to collect the supernatant. The absorbance values of 296 nm for CS-1 and 410 nm for Ce6 were measured for constructing standard concentration curve. The LC and EE were calculated using the following formulas: LC (%) = (Mt-Mu)/Mp × 100%, and EE (%) = (Mt-Mu)/Mt × 100%.
Where MT represents the total mass of CS-1 and Ce6 for loading, MU represents the unencapsulated mass and MP represents the mass of CP NPs.
To monitor CS-1 release behavior, 2 mg CPCC NPs with 1.56 mg CS-1 were dispersed in PBS solution (1 mL) (pH7.4/6.8). The supernatant containing released CS-1 was collected at different time points by centrifuging at 8000 rpm for 10 min, simultaneously, the content of CS-1 was detected by UV–Vis spectrophotometer. For the group treated with NIR radiation, the suspended materials were radiated (660 nm, 0.2 W/cm2, 5 min) with 3 lasers on/off cycles.
Biosafety and biocompatibility assay
The hemolysis assay was performed using the whole blood of healthy female BALB/c mice. Blood samples were centrifuged at 3000 rpm at 4 °C for 5 min and washed 3 times with PBS. 50 μL of 4% erythrocytes (v/v) was dissolved in 950 μL PBS (pH 7.4) with CP, CPCC, and CPCCM NPs (concentrations varied from 25 to 300 μg/mL) and incubated at 37 °C for 4 h. All samples were centrifuged at 3000 rpm at 4 °C for 5 min. The absorbance values (540 nm) of supernatants were measured on the UV–Vis spectrophotometer. Hemolysis rates were calculated using Eq. (1). In parralle, the separated erythrocytes were used for morphological imaging under microscope.
$${\text{Hemolysis}}\,{\text{(\% ) = }}\left( {{{\text{I}} \mathord{\left/ {\vphantom {{\text{I}} {{\text{I}}_{0} }}} \right. \kern-\nulldelimiterspace} {{\text{I}}_{0} }}} \right) \times 100\%$$
(1)
where I means the absorbance of supernatant containing erythrocyte suspension and NPs and I0 means the absorbance of the whole erythrocytes-lysed water.
Platelet aggregation assay: Platelet-rich plasma prepared from the whole blood of BALB/c mice was mixed with CP, CPCC, CPCCM NPs (50 μg/mL), or thrombin (5 μg/mL) solution. After incubation for 1 h at 37 °C, the absorbance values of 650 nm were measured. Meanwhile, the samples containing plasma and thrombin or PBS (9:1, v/v) were used as positive and negative controls, respectively.
Cytotoxicity assay. NIH-3T3 and MDA-MB-231 cells were cultured in the 96-well plate (5 × 104 cells/ well) for 24 h. Then, CP, CPCC, and CPCCM NPs with different concentrations (25 to 200 μg/mL) were incubated with cells for 24 h before the addition of 100 μl MTT (0.5 mg/mL). After 4 h, MTT was replaced by DMSO. The absorbance values of cells at 490 nm were measured for cell viability assay according to Eq. (2).
$${\text{Cell Viability (\% ) = }}\left( {{{{\text{OD}}_{{490\,{\text{nm/sample}}}} } \mathord{\left/ {\vphantom {{{\text{OD}}_{{490\,{\text{nm/sample}}}} } {{\text{OD}}_{{490\,{\text{nm/control}}}} }}} \right. \kern-\nulldelimiterspace} {{\text{OD}}_{{490\,{\text{nm/control}}}} }}} \right) \times 100\%$$
(2)
Functional test of erythrocyte-cancer hybrid membrane
CLSM was used to evaluate the targeting ability of CPCC@Lip, CPCC@231, and CPCC@RBC-231. Membrane-coated CPCCM NPs (Ce6: Ex/Em = 400/653 nm) were used to estimate the cellular uptake efficacy in vitro. SMC and MDA-MB-231 cells were cultured in the 6-well plates with cover slip (1 × 106 cells/well), respectively. After 24 h, PBS washed cells were separately incubated with CPCC@Lip, CPCC@231, and CPCC@RBC-231 NPs (50 μg/mL) for 6 h, respectively. Then, cells were washed with PBS before CLSM imaging. The immune evasion assay was performed with the above method by using RAW264.7 cells as target cells.
Anti-tumor performance in vitro
Cell uptake assay: MDA-MB-231 cells were cultured into 12-well plates with cover-slip (5 × 104 cells/well) for 24 h. The medium was then replaced with fresh medium containing CPCCM NPs (equivalent to 5 μg/mL Ce6) and incubated for 2, 4, 6, and 8 h. The nuclei were stained with Hoechst 33342 and images of cells were captured under CLSM.
Cell viability assay: MDA-MB-231 cells seeded in the 96-well plate (5 × 103 cells/well) and cultured for 24 h. The hypoxic environment was constructed according to the instructions of the Anaero Pack-Anaero kit. Then, different materials were incubated with cells for 24 h before the addition of 100 μL of MTT (0.5 mg/mL). After 4 h, excess MTT was replaced by the DMSO and the absorbance values of samples were measured at 490 nm.
Live/dead staining: The hypoxic environment was constructed according to the instructions of the Anaero Pack-Anaero kit. The MDA-MB-231 cells were cultured into 12-well plates with cover-slip (5 × 104 cells/well) for 24 h before the medium containing PBS, CPM, CS-1, Ce6, CPCM, and CPCCM NPs (CP, 50 μg/mL; Ce6, 2.5 μg/mL; CS-1, 5 μg/mL) was substituted with fresh medium. The cells were irradiated with 660 nm laser (0.2 W/cm2) for 3 min. After treating for 48 h, the MDA-MB-231 cells were stained with Calcein AM/Propidium Iodide for 5 min for imaging under CLSM.
ROS assay: MDA-MB-231 cells in 12-well plates with round cover slip (5 × 104 cells/well) were cultured for 24 h. Then, the medium was replaced by the fresh medium containing PBS, CPM, CS-1, Ce6, CPCM, or CPCCM (CP, 50 μg/mL; Ce6, 2.5 μg/mL; CS-1, 5 μg/mL). After 6 h, the cells were irradiated with 660 nm laser (0.2 W/cm2) for 3 min, followed by the addition of DCFH-DA. The hypoxic environment was constructed according to the instructions of the Anaero Pack-Anaero kit. MDA-MB-231 cells washed with PBS were imaged under CLSM 30 min later.
For 1O2 generation and PDT enhancement assay: MDA-MB-231 cells were seeded into 12-well plates with cover slip (5 × 104 cells/well) and incubated with PBS, CPM, CS-1, Ce6, CPCM or CPCCM (CP, 50 μg/mL; Ce6, 2.5 μg/mL; CS-1, 5 μg/mL) for 6 h. Then, cells were exposed to 660 nm laser irradiation for 3 min (0.2 W/cm2). Cyto-ID® Hypoxia/Oxidative Stress Detection regent was added to the plates and incubated for 0.5 h for confocal imaging. The fluorescence signal intensities of the hypoxia probe (Ex/Em = 590/670 nm), and ROS probe (Ex/Em = 490/525 nm) and Hoechst 33,342 (Ex/Em = 346/460 nm) in the cells was measured using spectrofluorometer.
Tumor mouse model
For the orthotopic breast cancer model, 100 μL PBS/Matrigel mixture containing 1 × 107 MDA-MB-231 cells was injected into the fourth inguinal mammary fat pad on the left of healthy female BALB/c nude mice (4–6 weeks old). Tumor size was measured with caliper and tumor volume was calculated by the standard formula 0.5 × L × W2, where L is the long diameter and W is the short diameter.
Body imaging and biodistribution of CPCCM NPs
200 μL free Ce6, CPCC, and CPCCM NPs (2.5 mg/kg Ce6 equivalent) solutions were injected intravenously into the female BALB/c mice (n = 3 per group). Blood samples were obtained from the retro-orbital plexus of mice at different time intervals (0.1, 1, 2, 4, 6, 8, 12, and 24 h). The blood sample was centrifuged at 3500 rpm for 10 min at 4 °C to obtain the plasma. 100 μL plasma from each sample was imaged using IVIS kinetics optical system (PerkinElmer, CA) to illustrate the fluorescence signal of Ce6.
For distribution of CPCCM in vivo: 14 days after orthotopic tumor implantation, mice were intravenously injected with equal volumes of free Ce6 or Ce6-labeled CPCC and CPCCM NPs (5 mg/kg of Ce6) (n = 3 per group). The whole bodies of mice were imaged at different time points using IVIS kinetics optical system. The major organs (heart, liver, spleen, lung, and kidney) and tumors were collected 48 h post-administration for imaging immediately under the same system.
Anti-tumor performance in vivo
The healthy female BALB/c nude mice (4–6 weeks old) were subcutaneously injected with MDA-MB-231/Luc tumor cells, as described above. When the tumor volumes reached 50 mm3, mice were randomly divided into PBS group, CS-1 group, Ce6 + L group, CPCM + L group, and CPCCM + L treatment group (n = 5 per group). Mice were injected intravenously with the same doses of Ce6 (2.5 mg/kg) or CS-1 (1 mg/kg). 4 h later, the mice were locally irradiated with 660 nm laser (0.2 W/cm2) for 5 min. The body weights and tumor volumes of mice were monitored and recorded on the day of 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20. The primary tumor of each mouse was detected by bioluminescence imaging on the 20th day. The collected tumors and main organs were fixed in 4% paraformaldehyde for H&E and TUNEL staining. Meanwhile, blood samples of each group were collected for biochemical and hematological assay.
Antitumor invasion and metastasis in vivo
4T1/Luc tumor model was established by subcutaneously injecting tumor cells into the female BALB/c mice (normal immune system, 4–6 weeks old). When the tumor volumes reached 50 mm3, they were randomly divided into PBS group, CS-1 group, Ce6 + L group, CPCM + L group, and CPCCM + L treatment group. They were intravenously injected with the same doses of Ce6 (2.5 mg/kg) or CS-1 (1 mg/kg) and with or without local laser irradiation (660 nm, 0.2 W/cm2, 5 min) 4 h after injection. After 20 days, the drug administration was stopped and the change in tumor volumes was observed. Tumors and main organs were collected for H&E and immuno-histochemical (IHC) staining using HIF-1α and MMP-9 antibodies. The total number of metastatic nodules in the lung and liver was counted to evaluate the anti-metastatic effects of CPCCM (n = 3 per group).
Statistical analysis
Data were expressed as mean ± standard deviation (SD). T-test or One-way (ANOVA) test by using the GraphPad Prism 8.0.2 software was applied to test the significance between groups, where significant differences were defined as *p < 0.05, **p < 0.01, ***p < 0.001.