Cell lines and materials
The magnetotactic bacterium Magnetospirillum gryphiswaldense MSR-1 is stocked in our laboratory. Cell lines SK-BR-3 (ATCC#HTB-30) and MDA-MB-468 (ATCC༃HTB-132) were kindly donated by National Center for Nanoscience and Technology. The anti-HER2 humanized mAb trastuzumab (TZ) was from Roche (Roche, Switzerland). McCoy's 5A medium, Leibovitz's L-15 medium, fetal bovine serum (FBS), and rabbit anti-HER2 Ab were from Thermo Fisher (Waltham, MA, USA). Human IgG was from Sigma-Aldrich (Germany). 4’,6’-diamidino-2-phenylindole dihydrochloride (DAPI), 1,1-dioctadecyl-3,3,3,3- tetramethylindocarbocyanine perchlorate (DiI), Prussian Blue Iron Stain Kit, and Enhanced BCA Protein Assay Kit were from Solarbio Science &Technology (Beijing, China). Goat anti-rabbit IgG-HRP, rabbit anti-mouse IgG-HRP, rabbit anti-goat IgG-HRP, Peroxidase-AffiniPure F(ab')2 Fragment, and rabbit anti-goat IgG (H + L) were from Shanghai Universal Biotech Co.
Blood samples were taken from 10 healthy volunteer donors after obtaining informed consent for blood collection and subsequent analysis, and stored in 10-ml tubes containing K2 EDTA to prevent clotting. Samples were centrifuged (1300 x g, 15 min, 4°C) to remove red and white cells, and supernatants were collected and stored at -80°C for subsequent experiments.
M. gryphiswaldense MSR-1 were cultured in a 7.5-L fermentor (BioFlo 110; New Brunswick Scientific; CT, USA). Inoculum was cultured in sodium lactate medium as described in our 2008 report . Cells was harvested at 3000 rpm for 30 min at 4°C. BMPs were extracted as described in our 2019 report . In brief, harvested cells were resuspended in 10 mM PBS (10 ml per g bacterial pellet; pH 7.4) and disrupted by ultrasonication (150 W, 30 min) (model JY92-IIN; Scientz; Xiamen, China) on ice. BMPs were collected from solution by magnet at 4°C overnight, supernatant was discarded, and precipitate was resuspended in PBS. The above steps were repeated until protein in supernatant showed no decrease. BMPs were treated with 1 mg/ml proteinase K for 3 h at 56°C, electroeluted as described in our 2011 report , suspended in PBS at final concentration 1 mg/ml, and sterilized by cobalt-60.
Purification of recombinant protein A
Nucleotides of protein A (Z domain) were optimized using a JAVA adaptation tool for soluble expression in E. coli BL21. Recombinant protein containing linker peptides and cysteine was synthesized at N-terminus, cloned into pET28(a+) plasmid with kanamycin-resistant gene, and transformed into E. coli BL21. The protein expressed by recombinant gene was termed RA. E. coli BL21 with plasmid was cultured in LB medium, expression was induced by addition over 8 h of isopropyl β-D-thiogalactopyranoside (IPTG) at final concentration 1 mM. Cells were collected by centrifugation (10,000 x g, 10 min), resuspended in 10 mM PBS (10 ml per g bacterial pellet; pH 7.4), disrupted by sonication at 30% amplitude (200 W, 30 min) on ice, and centrifuged again (10,000 x g, 30 min). RA protein present in supernatant was purified using HisTrap Fast Flow (FF) Crude Column as per manufacturer's instructions. Purified RA was freeze-dried and store at -20°C for subsequent experiments.
Identification of RA and its function
Purified RA was added with protein loading buffer (0.01 (w/v) bromophenol blue, 0.04 M dithiothreitol (DTT), 10% glycerol, 2% (w/v) SDS, 62.5 mM Tris-HCl, pH 6.8), boiled for 10 min at 100°C, and centrifuged. Supernatants were loaded onto 10% gels and run at 120 V for 1–2 h in a Mini-PROTEAN Tetra Electrophoresis System (Bio-Rad; USA). Gels were stained by Coomassie Brilliant Blue G250 for direct protein imaging, or transferred onto PVDF membrane in TransBlot SD Semi-Dry Electrophoretic Transfer Cell (Bio-Rad) for Western blotting. Membranes were blocked with 5% skim milk for 2 h at room temperature (RT), incubated with mouse anti-FLAG tag Ab (dilution 1:2000) for 8 h, washed 3x with PBST (PBS containing 0.05% Tween-20), incubated with HRP-labeled goat anti-mouse Ab for 2 h at RT, and washed 3x with PBST. Proteins on membranes were displayed using Immobilon Western chemiluminescent HRP substrate, and visualized using an imaging system (Fusion FX6; Vilber; France).
Enzyme-linked immunosorbent assay (ELISA) analysis of functions of RA and RA-decorated BMPs
The ability of RA to bind IgG was analyzed by ELISA. 2 µg RA in 100 µl carbonate-bicarbonate buffer (pH 9.6) was incubated on 96-well microtiter plate overnight at 4°C. Plate was washed 3x next day with PBST, added with 300 µl of 1% BSA (diluted in PBST) for 1 h at RT to block nonspecific binding sites, washed 3x with PBST, added with same number of moles of rabbit anti-mouse IgG-HRP and rabbit anti-goat Peroxidase-AffiniPure F(ab')2 Fragment for 1 h at RT, washed 3x with PBST, and added with 100 µl of 3,3′,5,5′-tetramethylbenzidine (TMB) solution. After 8 min, reaction was stopped by adding 50 µl of 2 M H2SO4. Absorbance at wavelength 450 nm was read by microtiter plate reader (ELx800; BioTek, U.S.). Samples without RA input were used as negative controls.
For BMP-RA, microtiter plate was blocked with 1% BSA overnight, washed 3x, added with 1 ng BMP-RA, placed over magnet for 5 min to isolate complex, added with rabbit anti-mouse IgG-HRP (1:20000 dilution in PBS) for 1 h at RT, and subsequent steps were the same as for RA. BMP wild-type (BMP-WT) was used as negative control.
For binding of BMP to human epidermal growth factor receptor-2 (HER2), initial processing of microtiter plate was the same as for BMP-RA. Then, plate was added with BMPs, washed, added for 1 h with HER2 at RT, washed 3x with PBST, added with 100 µl rabbit anti-HER2 Ab, incubated for 1 h with BMPs at RT, washed 3x, added with goat anti-rabbit IgG-HRP for 1 h, and subsequent steps were the same as for BMP-RA.
Preparation of functional BMPs
TZ functionalized BMPs were prepared using N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) and glutaraldehyde as cross-linkers, and termed BMP-RA-TZ and BMP-GA-TZ respectively. For BMP-RA preparation, 1 mg BMP was resuspended in HEPES (10 mM, pH 8.0) containing 1 mM SPDP, solution was ultrasonicated for 1 min followed by 1-min intervals (this step was repeated 30x), and mixture was washed 3x with HEPES. RA (1 mg/ml) was suspended in 10 mM HEPES, ultrasonicated for 1 min followed by 1-min intervals (repeated 30x), supernatant was removed and washed 3x with HEPES under magnet, and resuspended in HEPES. The BMP-complex was termed BMP-RA. SDS-PAGE was used to confirm RA linkage on magnetosome, 0.2 mg BMP-RA was loaded, and subsequent steps were the same as for RA. For BMP-RA-TZ preparation, TZ solution (500 µg/ml) was suspended with modified BMPs and incubated for 2 h at 37°C with rotation (200 rpm). BMP-RA-TZ was washed 6x with HEPES and stored at 4°C. BMP-GA-TZ was prepared as described in our 2019 report . For confocal laser scanning microscopy (CLSM) and flow cytometry (fluorescence activated cell sorting; FACS), 1 mg BMP was FITC-labeled by mixing with 300 µl of 1 mg/ml FITC and incubated overnight at 4°C with shaking.
BMP samples were incubated with human plasma or IgG-enriched human plasma for 2 h at 37°C with shaking (200 rpm), and washed 6x with HEPES. For IgG-enriched human plasma experiments, 1 ml human normal plasma was added with 6 mg IgG. Following the last washing step, BMPs were resuspended with 1 ml HEPES in a 1.5-ml Eppendorf tube.
Characterization of BMPs
Transmission electron microscopy (TEM): BMP sample (0.2 mg) was suspended in 1.5-ml Eppendorf tube with 1 ml double distilled water (ddH2O), ultrasonicated for 1 min, and BMPs were collected under magnet and resuspended in 1 ml ddH2O. These steps were repeated 3x. 10 µL of this suspension was dropped onto copper grid and air dried for 10 min. Samples were observed by TEM (model JEM-1230; JEOL; Tokyo, Japan).
Zeta-potential and DLS measurement: BMP sample was resuspended in ddH2O at concentration 0.01 mg/ml and ultrasonicated for 10 min. Samples were combined in sample pool and measured using Zetasizer Nano ZS particle size analyzer (Malvern Instruments; Worcestershire, UK) at 25°C.
Immediately following the last washing step, defined amount of BMP-corona complex (0.2 mg) was resuspended in protein loading buffer, boiled for 10 min at 100°C, BMPs were removed by centrifugation (12,000 x g, 20 min), and equal sample volume was loaded on 10% gel. SDS-PAGE was performed at 80 V for 30 min, switched to 120 V for ~ 90 min until bromophenol blue neared the gel end, and gel was stained with protein silver stain kit.
Quantitative analysis of TZ on BMPs
TZ supernatant before and after coupling to BMP was stored in advance, and analyzed using Enhanced BCA Protein Assay Kit as per manufacturer's protocol. Standard curve was constructed based on eight BSA concentration points (0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5 mg/ml). 20 µl samples or BSA (control) were placed on 96-well plates, added with 200 µl BCA working solution, mixed thoroughly, incubated for 30 min at 37°C, and read by microplate spectrophotometer (Power Wave XS2; BioTek) at 562 nm. Sample concentrations were calculated based on the standard curve. Amount of TZ coupled to BMP was calculated as TZ amount before coupling -TZ amount TZ after coupling.
Protein identification and classification
BMP-corona complexes were obtained by the above methods, and proteins from SDS-PAGE 10% gels were excised and in-gel trypsin-digested by the method of Shevchenko et al. . In brief, 100 µg protein was dissolved in 50 mM ammonium bicarbonate (ABC) solution, reduced with DTT for 45 min at 56°C, alkylated with iodoacetamide (IAM) for 30 min at RT in the dark, and the solution was transferred to 10K ultrafiltration tube (Vivacon 500; Sartorius, USA) and centrifuged (14000 x g, 20 min). Proteins were washed with 50 mM ABC, added with 2 µg trypsin in 50 µl ABC, and incubated overnight at 37°C. The ultrafiltration tube was centrifuged (14000 x g, 20 min), digested peptides were transferred to a collection tube, and ABC was added to ultrafiltration tube to wash digested peptides into collection tube. Collected solution was diluted with 0.1% formic acid (FA) for nano-LC-MS analysis. NanoAcquity nano-HPLC (Waters Corp.; Milford, MA, USA) was used for nano-LC separation. Trap column was Thermo Acclaim PepMap 100 (75 µm ⋅ 2 mm, C18, 3 µm), and custom-made analytical column was fused silica capillary (I.D. 100 µm; Polymicro Technologies) filled with 20 cm stationary phase (Aqua 3-µm C18 125A; Phenomenex). Gradient elution program: mobile phase increased linearly from 1% B to 35% B in 65 min. Mobile phase A was 0.1% FA in water; mobile phase B was 0.1% FA in acetonitrile. Protein false discovery rate was set at 4%, maximum protein mass was set at 600 kD, and generated peptide masses were compared to a reviewed human protein sequence database downloaded from UniProt (www.uniprot.org). Identification of a peptide required at least two assigned fragments; identification of a protein required at least two assigned peptides and five assigned fragments.
Targeted cell uptake evaluation of BMPs by flow cytometry and CLSM
To evaluate uptake of BMP-RA-TZ or BMP-GA-TZ by SK-BR-3 following incubation with human plasma or IgG-enriched human plasma, ~1⋅105 cells in 1 ml McCoy's 5A medium with 10% FBS were seeded in a 12-well plate, cultured for 24 h under standard conditions, and then incubated with various samples (BMP-RA-TZ, BMP-RA-TZ-plasma, BMP-RA-TZ-IgG plasma, BMP-GA-TZ, BMP-GA-TZ-plasma, BMP-GA-TZ-IgG plasma) of BMPs (10 µg/ml) in McCoy's 5A (0% FBS) for 2 h. For analysis of BMP uptake at various plasma concentrations, 1⋅105 cells were placed in McCoy's 5A with 0%, 10%, or 100% human plasma, incubated with BMPs at indicated concentrations for 2 h, and washed 2x with 10 mM PBS. For flow cytometric measurements, cells were treated with 0.5% trypsin-EDTA, collected by centrifugation, washed 3x with PBS (10 mM), resuspended in 400 µl PBS, and FITC fluorescence was measured with a flow cytometer (FACS Calibur; BD Biosciences; San Jose, CA, USA). Cells without BMP were used as negative control, and percentage of FITC-positive cells was calculated relative to negative control value. For CLSM, cells were seeded in a 24-well plate with cell slide, incubated with BMPs, washed 2x with 10 mM PBS,stained with 200 µl of 10 µM 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate for 10 min at 37°C, fixed with 4% polyoxymethylene (POM) for 10 min at RT, and stained with 200 µl of 10 µg/ml 4,6-diamidino-2-phenylindole dihydrochloride for 10 min at 37°C. Images showing cellular uptake and distribution of BMPs with FITC-fluorescence signals were obtained using an inverted CLSM instrument (model A1; Nikon, Japan). Uptake efficiency of MDA-MB-468 was evaluated by an analogous series of steps.
HER2 expression in SK-BR-3 and MDA-MB-468 cells
HER2 expression on SK-BR-3 and MDA-MB-468 cell surfaces was evaluated by CLSM and Western blotting. For CLSM, cells were seeded in 24-well plate with cell slide, incubated for 24 h, fixed with 4% POM for 10 min at RT, incubated with normal goat serum (Solarbio) for 1 h at RT, incubated with rabbit anti-HER2 Ab (1:500 v/v, in 0.01 M PBS; Invitrogen; USA) at 4°C overnight, incubated with Alexa Fluor 488-conjugated goat-anti-rabbit IgG (1:1000 v/v, in 0.01 M PBS) for 1 h in the dark, and stained with 10 µg/ml DAPI in the dark.
For Western blotting, cells were collected from incubated on 6-well plate, washed with PBS, and added with 250 µl native lysis buffer (Solarbio) on ice for 10 min. Supernatant was mixed with SDS-PAGE loading buffer, boiled at 100°C for 10 min, centrifuged, loaded on 10% gel, transferred to PVDF membrane, and blocked with 5% BSA. Membrane was incubated with rabbit anti-HER2 Ab (1:1000 v/v, in 0.01 M PBS) overnight at 4°C, washed 3x with TBST (PBS containing 0.05% v/v Tween 20), incubated with HRP-labeled goat anti-rabbit (1:20000 v/v, in 0.01 M PBS) for 2 h at RT, and imaged using an imaging system (Fusion FX6; Vilber).
Prussian blue staining
Cells were seeded in 24-well plate with cell slide, cultured for 24 h under standard condition, incubated with 10 µg/ml BMPs for 2 h, washed 3x, fixed with 4% POM for 10 min at RT, the iron was detected by Prussian blue staining; i.e., equal volumes of Perls stain A and B were mixed, added to fixed cells and tissue sections, and incubated for 30 min at 37°C. Samples were then washed 3x with Millipore water, immersed in Nuclear Fast Red solution for 2 min, cell slide was washed 3x with Millipore water, and cellular uptake and distribution of BMPs were imaged using a Zeiss microscope (Axio Cam MR3; Germany).
Statistical analysis was performed using student t-test for two groups, and one-way ANOVA for multiple groups. All data are given as mean ± standard deviation (S.D.). Difference are considered statistically significant as *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.