AR-N 7520 electron beam resistor and AR 300-46 development were purchased from Allresist (Germany). Fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM) high glucose, RPMI-1640 medium, trypsin-EDTA 0.25%, and penicillin-streptomycin were purchased from Biological Industries (Kibbutz Beit-Haemek, Israel). Cell Count Kit-8 (CCK8) was purchased from MedChemExpress (USA). TRITC Phalloidin and DAPI were purchased from Soledad Bao Technology Co. (Beijing, China). L929 mouse fibroblasts, RAW 264.7 macrophages, HepG2 liver cancer cells were ordered from Chinese Academy of Sciences cell bank.
Manufacture of nanobots
The manufacturing process of the nanorobots is shown in Figure 1. First, a sacrificial layer of aluminum (Al) of 30 nm was evaporated on a silicon wafer with a deposition rate of 6 Å/s. Next, a 100 nm layer of electron beam resist (EB) (AR-N 7520.07) was deposited by spin coating with a coating speed of 3000 rpm, followed by baking at 95° C for 3 mins. Next, the sample was exposed to an electron beam exposure unit with an exposure meter of 750 u/cm2 to create L-shaped patterns as shown in Fig. 1a. After that, the sample was developed by inductively coupled plasma (ICP) etching for 20 s under 300 W to remove excess Al material not protected by the EB resist, as shown in Fig. 1b. Finally, the sample was coated with 20 nm silver (Ag), 70 nm nickel (Ni) and 10 nm titanium (Ti) layers by electron beam evaporation (EBE), as shown in Figure 1c; the coating rate for these three materials was 4 Å/s, 6 Å/s, and 6 Å/s, respectively.
The sample was placed in a mixed solution of sodium hydroxide (NaOH) and hydrogen peroxide (H2O2) at 65°C for 5 min to dissolve the sacrificial Al layer, as shown in Fig. 1d. The H2O2 will decompose under heating to produce bubbles to push the nanorobots out of the substrate, allowing the nanorobots to be dispersed in solution after release. The nanorobots were observed before immersion in NaOH and H2O2, after 10 min of submersion and after 20 min of submersion, as shown in Figs. 2a–c respectively. It was observed that the nanorobots were completely released and dispersed in the solution without aggregation after 20 min of submersion, as shown in Fig. 2c.
Cell cultures were maintained at 37°C, 5% CO2. L929 mouse fibroblasts and RAW 264.7 macrophages were cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. HepG2 liver cancer cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. The medium was changed every 48 h and the cells were subcultured with 0.25% trypsin-EDTA with a 1 min incubation at 37°C.
Cellular cytotoxicity was determined using a CCK-8 assay. Samples were sterilized by autoclaving at 121°C, 20 min. Cell suspension with a density of 2 × 104 cells per ml were seeded on the samples. The cytotoxicity of the cell was measured at different times according to the following steps. Cells incubated with samples were cleaned with PBS, then fresh complete DMEM medium containing 10% by volume CCK-8 was added. Two hours later, 100 μL solutions for each sample were transferred to a 96-well culture plate and the absorbance was measured at 450 nm by an enzymatic labeling instrument (Tecan).
Cell morphology by SEM and immunofluorescence
Cells were exposed to samples in a 48-well cell culture plate. After 24 h, cells were washed three times with phosphate buffered saline (PBS) and fixed with 4% paraformaldehyde for 15 min at room temperature, then washed with PBS. For SEM, cells were dehydrated in graded ethanol (30-100% v/v) and air-dried.
For immunofluorescence staining, cells were then permeabilized with 0.1% Triton X-100 for 15 min at room temperature, washed three times (5 min each) with PBS. Actin filaments were labeled with tetramethylrhodamine (TRITC)-labeled phalloidin for 40 min and cell nuclei were stained with DAPI for 5 min. Stained cells were washed and sealed with an anti-fluorescence quenching agent. Imaging was performed on the Nikon A1R confocal microscope.
Staining of live and dead cells
Staining of live and dead cells was performed using Hoechst 33258 assays and propidium iodide. Briefly, the cells were cultured on the specimens with a density of 1 × 106 cells per well. Hoechst 33258 and propidium iodide and calcium-AM were diluted to final concentrations of 10 μg/mL in PBS, respectively. After one day, 100 μL of the mixed solution was added to each sample and the cells were stained at 37°C for 10 min.