Bottom-up self-assembly of heterotrimeric nanoparticles and their secondary Janus generations† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c9sc02961c

Designed Janus silica nanoparticles can stimulate stronger phagocytosis and exhibit higher cargo transport across an in vitro epithelial monolayer model mimicking the human intestinal epithelium.

1 mL cold PBS was used to make cells detached from the dishes). Cell counting was performed by a hemocytometer after cell collection. Then cells were centrifuged again and the supernatant was removed. 120 μL cell lysis buffer was added to allow the cells to break apart in ultrasonic water bath. The pellets were collected by centrifugation at 13,000 rpm for 10 min. Aqueous NaOH solution (1 M) was then added to dissolve the pellets for 24 h. The silicon content in the final solutions were measured by ICP-OES, the result of which were then converted into silica amount.
Confocal fluorescence microscopy assays for actin staining 2 Each cell line was cultured on a sterile coverslip in a 6-well plate with a density of 2×10 5 cells per well. After culturing for 24 h, the J-SNPs or S-SNPs (100 μg/well, with concentration of 50 μg/mL) were further incubated with cells for 0.5, 2 and 4 h. At each time point, cell culture medium was removed, 1 mL warm PBS was used to gently wash off attached particles for twice.
The cells were fixed with 4% electron microscopy-grade paraformaldehyde (PFA) for 30 min.
Once fixed, the cells were washed with PBS and permeabilized with 0.1% Triton X-100 in PBS for 3 min. Then the cells were washed again with PBS and 2.5 units/mL rhodamine phalloidin was added to each well for 15 min to stain polymerized actin filaments. Each well was washed with PBS. The coverslips were taken out, stuck on glass slides and viewed at 63× magnification.
Bright-field and fluorescent images of cells were acquired and merged. The phagocytosis level was determined by the obtained red fluorescence intensity. To visually detect the fluorescent intensity coming from the activation of phagocytosis, the confocal microscopy parameters were set to offset the fluorescent signals from the control group (PBS treated sample) of each cell line.

Grafting of rhodamine-B-isothiocyanate (RITC) on silica nanoparticles
3 mg of RITC was reacted with 20 μL (3-aminopropyl)triethoxysilane (APTES) in 1 mL ethanol solution under dark condition for 24 h. Subsequently, 4 mg of the silica nanoparticles were reacted with the as prepared 1 mL RITC-APTES stock solution under dark conditions for 24 h.
The RITC grafted silica nanoparticles were collected by centrifugation and washed with ethanol several times to remove the free dye molecules. To normalize the RITC amount in each type of silica nanoparticles, ~1 mg RITC grafted silica nanoparticles were dissolved in NaOH solution aqueous (1 M). The RITC concentrations were then measured by UV-Vis spectroscopy. The RITC grafting density was measured to be 4.6 μg/mg and 5.3 μg/mg for J-SNPs and S-SNPs, respectively.

Cellular uptake of nanoparticles evaluated by confocal fluorescence microscopy
Each cell line was cultured on a sterile coverslip in a 6-well plate with a density of 2×10 5 cells per well. After culturing for 24 h, the RITC conjugated J-SNPs or S-SNPs (100 μg/well) were further incubated with cells for 4 h. The cells were fixed and the cell nuclei were stained by DAPI (blue fluorescence). The samples were observed under a fluorescence microscope (Leica SP8).

MTT assay
The potential biotoxicity of J-SNPs or S-SNPs were tested in RAW264.7, HCT116, CHO-K1 and KHOS cell lines. 8000 cells were seeded in each well of 96-well plates. After incubation for 24 h, the medium was replaced with 100 uL fresh medium. Then 100 uL of particle medium solution with different particle concentration were prepared and added to each well. After further incubation for 24 h and 72 h, 20 uL MTT PBS solution (5 mg/mL) were added to each well. Incubation for 4 h, then the culture medium was replaced with 200 uL dimethyl sulfoxide (DMSO). The related absorbance were recorded at 540 nm using a Tecan Infinite M200 PRO microplate reader.

Fluorescence-activated cell sorting (FACS) analysis of cytochalasin D (noted as Cyto D) inhibited cellular uptake in RAW264.7 cell line
The density of 1×10 5 cell/well and 12-well plates were used for seeding. The cell culture and sample preparation was the same with cellular uptake except without cell lysis. Before the incubation of SNPs with cells, Cyto D (10 μg/well) was introduced in the culture medium and incubated for 0.5 h at 37 o C. Afterwards, RITC conjugated J-SNPs and S-SNPs (50 μg/well) was added to the culture medium and incubated for another 0.5 h. Cells were collected and analysed by CytoFLEX flow cytometer. The results were reported as the mean fluorescence intensity obtained by measuring 10,000 cells between independent experiments. Error bars represent one standard deviation around the mean.

Scanning electron microscopy (SEM) observation of J-SNPs on cells
Cells were grown on Thermonox coverslips (Nunc, Roskilde, Denmark) and treated with particles for 1 h. After incubation, the medium was removed and the samples were gentle

Synthesis of MCM-48
MCM-48 mesoporous silica nanoparticles were synthesized according to a literature report. 1 In a typical synthesis, 500 mg of cetyltrimethylammonium bromide (CTAB) and 1 g of F127 were dissolved in an aqueous solution containing 96 mL distilled water, 10 g of ammonium hydroxide solution (28 wt%) and 34 g of ethanol. Afterwards, 1.8 g of TEOS was added into the above solution and vigorously stirred for 1 min. Then the mixture was kept under static solution for 24 h at room temperature.

Synthesis of dendritic mesoporous organosilica nanoparticles (DMSNs)
Dendritic mesoporous organosilica nanoparticles (DMSNs) were synthesized according to literature with slight modification. 2 At first, 380 mg of CTAB and 168 mg of sodium salicylate were added in 25 mL of water containing 68 mg of triethanolamine (TEA). Afterwards, 6 mL of TEOS was added to the above solution and vigorously stirred for 4 h under 80 °C.

Caco-2/M cell monolayer
An in vitro human intestine Caco-2/M cell model was cultured to investigate the transcellular uptake of J-SNPs and S-SNPs. The human intestine Caco-2/M cell model was obtained based on previous reported protocols. 3,4 Briefly, 500 µL culture medium containing 5×10 5    Further discussions: N 2 sorption-desorption analysis was conducted to characterize the porous structures ( Figure S3). The ABC type heterotrimeric nanoparticles showed a solid structure.
The nitrogen sorption isotherms of J-SNPs were type IV, typical of mesoporous materials. J-SNPs exhibited a pore size of 15.1 nm, a specific surface area of 97.6 m 2 ·g -1 , a pore volume of 0.33 cm 3 ·g -1 (Table S1).

Further discussions:
The RITC was chemically grafted on the silica nanoparticles ( Figure S6) and neglectable amount of rhodamine-B was released from the nanoparticles during the cell experiment ( Figure S7). Statistical analyses were performed by using two-way ANOVA. "ns" indicates not significant (p>0.05) and **** indicates significant difference (p < 0.0001).          to G-actin. 6 When phagocytosis is activated, G-actin polymerizes and forms F-actin.
Rhodamine phalloidin (noted as RH phalloidin, red fluorescence) has been widely used to specifically bind and stain F-actin rather than G-actin as an indicator of the activation of phagocytosis pathway. 7,8 The phagocytosis extent was determined by the intensity of the red fluorescence in each image. 9 As shown in Figure S21 After the burst phagocytosis is completed, F-action can easily dissociate into G-actin, 12 thus the red intensity decreased, consistent with a previous report. 13         Further discussion: It has been reported that the initiation of phagocytosis is determined by the local shape of the particles when contacting with cell membrane, whereas the contact angle plays a key role. 14 For example, rod-like particles can easily initiate phagocytosis at the tip side. 15 Oblate ellipsoids are also more efficiently internalized by macrophages than spherical ones. 16 In our case, compared to S-SNPs, the unique "dent" part of the J-SNPs is similar to an