Supporting Information

Transcription

1 Supporting Information Microfluidic Electroporation-Facilitated Synthesis of Erythrocyte Membrane-Coated Magnetic Nanoparticles for Enhanced Imaging-Guided Cancer Therapy Lang Rao, Bo Cai, Lin-Lin Bu, Qing-Quan Liao, Shi-Shang Guo, Xing-Zhong Zhao, Wen-Fei Dong,*, and Wei Liu*, Key Laboratory of Artificial Micro- and Nano-Structures of Ministry of Education, School of Physics and Technology, Wuhan University, Wuhan, Hubei , China Department of Oral Maxillofacial Head Neck Oncology, School and Hospital of Stomatology, Wuhan University, Wuhan, Hubei , China Key Laboratory of Bio-Medical Diagnostics, Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou, Jiangsu , China * Corresponding author wliu@whu.edu.cn. and wenfeidong@126.com. S1

2 Experimental Details Cell and Animal Models. MCF-7 human breast cancer cells and RAW murine macrophage-like cells were provided by Zhongnan Hospital of Wuhan University and College of Life Science of Wuhan University. Two kinds of cells were cultured in Dulbecco s modified Eagle s medium (DMEM; high glocuse, Hyclone, USA) supplemented with 10% fetal bovine serum (FBS; Hyclone, USA) and 1% penicillin and streptomycin at 37 C and 5% CO 2 atmosphere in a cell incubator (Thermo Forma Series II, Thermo Scientific, USA). ICR mice (male, 6-8 weeks) and BALB/c nude mice (female, 6-8 weeks) were purchased from Hunan Silaike Jinda Laboratory Animal Co., Ltd. (China). BALB/c nude mice bearing MCF-7 breast tumor xenografts were obtained by subcutaneous (s.c.) injection of 50 µl serum-free cell medium containing MCF-7 cells into the shoulder of each mice. After the tumor volume reached ~50 mm 3, the tumor bearing mice were used for further experiments. All the animal procedures complied with the guidelines of the Institutional Animal Care and Use Committee at Wuhan University. Synthesis and Characterization of MNs. Clustered Fe 3 O 4 magnetic nanoparticles (MNs) were synthesized by a solvothermal reaction with slight modifications g FeCl 3 6H 2 O, 1.5 g NaOAc, 0.3 g C 8 H 5 Na 3 O 7 2H 2 O, and 1.0 g a,ω-bis{2-[(3-carboxy-1-oxopropyl)amino] ethyl poly(ethylene glycol) (PEG; M = 4000) were first dissolved in 40 ml ethylene glycol. Then the resulting mixture was stirred for 30 min and then sealed in a 50 ml teflonlined stainless-steel autoclave. The autoclave was heated to and maintained at 200 ºC for 8 h, and cooled to room temperature. The obtained solution was washed several times with ethanol and DI water, and harvested by using a magnet. The final product MNs were re-dispersed in DI water and stored at 4 ºC for future use. The obtained MNs were characterized by using a X-ray diffractometer (XRD; D8 Advance, AXS Instruments, Germany) and a scanning electron microscope (SEM; 6700F, JEOL, Japan). Preparation and Characterization of RBC-Vesicles. ICR mice was used for collecting RBCs to prepare RBC membranes. The whole blood was collected from the orbit of mice with the addition of S2

3 1.5 mg of ethylenediaminetetraacetic-acid (EDTA) per ml of blood for anticoagulation. The blood was centrifuged at 800 g for 5 min at 4 C to remove the plasma and the buffy coat. The resulting RBCs were washed with ice cold 1 phosphate buffer solution (PBS) for three times. Then, 0.25 PBS was added for hemolysis via a hypotonic medium treatment in an ice bath for 20 min. The released hemoglobin was removed by centrifugation at 4,000 g for 5 min, and the empty RBCs with a light pink color were collected and washed with 1 PBS for three times. The preparation processes were monitored using a conventional microscope (IX71, Olympus, Japan). Then, the obtained solution was sonicated for 5 min by using a bath sonicator at a frequency of 53 khz and a power of 100 W, and then extruded sequentially through a 400-nm polycarbonate porous membrane on a mini extruder (Avanti Polar Lipids, USA). The preparation of RBC membrane-derived vesicles (RBCvesicles) was monitored by measuring the hydrodynamic diameter and zeta potential of products after each step, using a dynamic light scatter (DLS; Nano-Zen 3600, Malvern Instruments, UK). Preparation and Characterization of RBC-MNs-C. To coat RBC membranes onto the MNs surface, 1 ml PBS containing 0.2 mg MNs was mixed with RBC-vesicles derived from 0.04 ml of mice blood. The mixture was subsequently extruded 11 times through a 200 nm polycarbonate porous membrane on an Avanti mini extruder, and then excess RBC-vesicles were removed by using a magnet. Finally, the newly prepared RBC-MNs were left in 1 PBS buffer overnight at 4 ºC for further use. The morphology of nanoparticles was characterized using a TEM (JEM-2010HT, Japan). The TEM samples were treated as described above. Stability experiments of nanoparticles were carried out by measuring the MNs and RBC-MNs-C in 100% FBS for 24 h with DLS. In Vitro Immune Escape Evaluation. First, MCF-7 cells were cultured in 12-well culture plates for 12 h. Then different concentrations of MNs, RBC-MNs-C, and RBC-MNs-E (i.e., 25, 50, and 100 µg Fe ml -1 ) were added to the culture medium, and the cells grown without the addition of nanoparticles were used as a control. The cells were further incubated for 4 h and washed. To quantify Fe uptake by cancer cells, the cells were treated as described above for Fe content S3

4 quantification using an ICP-AES (Iris Intrepid II XSP, Thermo Elemental, USA). Also, to investigate the influence of cell incubation time on the nanoparticle uptake, the nanoparticle concentration was fixed at 100 µg Fe ml -1, the incubation duration (i.e., 1, 2, 4, 8, 16, and 24 h) was changed, and the followed steps were processed as described above. In Vivo Toxicity Evaluation. For conducting systematic toxicity evaluation, 12 ICR mice (n = 6) were i.v. injected with 200 µl PBS or PBS containing 5 mg ml -1 RBC-MNs-E. In order to evaluate the general status of mice, veterinarian examined the mice every day and measured the body weights of mice every three day by using a digital scale. All mice were treated with euthanasia on the 30th d after the injection and their blood samples and major organs were collected for blood test and histology analysis. The blood parameters from the control and treated mice were detected by using a blood biochemical auto-analyzer (7080, HITACHI, Japan). The major organs were orderly fixed in 4% neutral formalin buffer, treated into paraffin, sectioned at 4 µm, stained with hematoxylin and eosin (H&E), and finally observed by using a typical optical microscope (BX51, Olympus, Japan). S4

5 Supplementary Figures Figure S1. (a) XRD spectrum and (b) SEM image of clustered Fe 3 O 4 MNs. S5

6 Figure S2. Bright field microscopy images of (a) RBCs and (b) RBC ghosts. S6

7 Figure S3. (a) Mean diameter and (b) zeta potential of RBC-vesicles following RBC ghosts derivation, sonication for 5 min and extrusion through 400- and 200-nm polycarbonate porous membranes. S7

8 Figure S4. Mean diameter of MNs and RBC-MNs in 100% FBS over 24 h. S8

9 Figure S5. TEM image of uncoated MNs. S9

10 Figure S6. TEM image of RBC-MNs-C. S10

11 Figure S7. Nanoparticle uptake by RAW macrophage-like cells at different incubated concentration with an incubation time of 4 h. S11

12 Figure S8. Nanoparticle uptake by MCF-7 cancer cells (a) with different incubation time at a concentration of 100 µg Fe ml -1 and (b) at different concentration with an incubation time of 4 h. S12

13 Figure S9. MCF-7 cancer cell viability after 24-h incubation with various nanoparticles at different concentrations. The cells grown without nanoparticles are used as a control. S13

14 Figure S10. BALB/c nude mice body weight change curves after cancer therapy. S14

15 Figure S11. ICR mice body weight change curves after injection of PBS or PBS containing RBC- MNs-E. S15

16 Figure S12. H&E-stained tissue slice photos of major organs from the euthanized mice on the 30th d after being i.v. injected with PBS or PBS containing RBC-MNs-E. S16

17 Figure S13. Blood biochemistry test: (a) AST: aspartate aminotransferase; ALP: alkaline phosphatase; ALT: alanine aminotransferase; (b) BUN: blood urea nitrogen; and (c) CRE: creatinine. And Complete blood panel analysis: (d) WBC: white blood cell; (e) RBC: red blood cell; (f) PLT: platelet; (g) HGB: hemoglobin; (h) HCT: hematocrit; (i) MCV: mean corpuscular volume; (j) MCH: mean corpuscular hemoglobin; (k) MCHC: mean corpuscular hemoglobin concentration. S17