Nanoparticles modulate autophagic effect in a dispersity-dependent manner

Autophagy plays a key role in human health and disease, especially in cancer and neurodegeneration. Many autophagy regulators are developed for therapy. Diverse nanomaterials have been reported to induce autophagy. However, the underlying mechanisms and universal rules remain unclear. Here, for the first time, we show a reliable and general mechanism by which nanoparticles induce autophagy and then successfully modulate autophagy via tuning their dispersity. Various well-designed univariate experiments demonstrate that nanomaterials induce autophagy in a dispersity-dependent manner. Aggregated nanoparticles induce significant autophagic effect in comparison with well-dispersed nanoparticles. As the highly stable nanoparticles may block autophagic degradation in autolysosomes, endocytosis and intracellular accumulation of nanoparticles can be responsible for this interesting phenomenon. Our results suggest dispersity-dependent autophagic effect as a common cellular response to nanoparticles, reveal the relationship between properties of nanoparticles and autophagy, and offer a new alternative way to modulate autophagy.

Finally, hexane was evaporated off, resulting in a semisolid iron oleate.

Synthesis of iron oxide nanoparticles
10 nm and 30 nm iron oxide nanoparticles (IO nanoparticles) were synthesized according to Park at el 1 . Briefly, 18 g iron oleate and 2.9 g oleic acid (90%, Alfa Aesar) was dissolved in 100 g 1-octadecene (90%, Alfa Aesar). The solution was degased at 100 °C for 60 min, and then heated to 320 °C with a rate of 3-4 °C/min. After refluxed for 60 min, the solution turned brown or black. Refluxed another 30 min, and cooled to room temperature. Nanoparticles were precipitated by adding ethanol and separated by centrifugation (5,000 x g, 3 min). The resulting IO nanoparticles were purified by re-dispersed in hexane and then precipitated in ethanol.
For synthesis of 10 nm IO nanoparticles (IO-10), 12.6 g iron oleate and 1.9 g oleic acid were dissolved in 60 g 1-octadecene. The solution was degased at 100 °C for 60 min, and then heated to 320 °C with a rate of 3-4 °C/min. Refluxed for 50 min and cooled to room temperature. Separated and purified as above.
Note: Refer above.

Synthesis of 100 nm iron oxide nanoparticles coated with sodium citrate
The 100 nm iron oxide nanoparticles (IO-100) were synthesized according to Liu  μm sterile filter before used.

Synthesis of gold nanoparticles
Gold nanoparticles were synthesized according to Turkevich method modified by

Determine the concentration of gold nanoparticles
We determined the concentration of gold nanoparticles by ICP-AES. Gold nanoparticles were digested by aqua regia at 100 °C for 1 hour and continued for 12 h to decompose HNO 3 . The residue was dissolved in 5% HCl for ICP-AES detection.
All concentrations of gold nanoparticles were expressed as the concentration of gold element.

Determine the concentration of silica nanoparticles
We (DMEM). All the cells were maintained at 37 °C, 5% CO 2 (Thermo forma series II).
When cells were 70% -90% confluent next day, added the mixture of

Construction of HeLa cell lines stably express GFP-LC3 (HeLa-GFP-LC3)
Lentivirus is highly efficient to integrate exogenous gene into host cells 5

Immunofluorescence staining
We performed immunofluorescence staining mainly according to the protocol from Cell Signaling Technology. Cells were seeded in glass-bottom (0.14 -0.18 mm) culture dish (Nest) so that they could be observed in oil immersion objective. Images were acquired using laser scanning confocal microscope (Leica TCS SP5). Measurement temperature was set as equal to or slightly higher than the temperature of solvent at room temperature. To measure the stability of colloid nanoparticles ( hydrodynamic diameter over time), measurement number was set to 30 or higher.

Scanning electron microscopy (SEM) imaging of cells
Results of size measurement were presented as Z-Average and polydispersity index (PdI). Z-Average is average diameter of intensity-weight distribution. Note: It is very important to use highly pure water and cleaning pipette.
For zeta potential measurement, IO nanoparticles were dispersed in 2 mM phosphate buffer with various pH (varied ratio of 2 mM monosodium phosphate and 2 mM disodium phosphate). The pH of silica nanoparticles was adjusted by HCl and NaOH.

Flow cytometry measurement
Cells were collected by trypsin after treatment and staining. After that, cells were placed in ice bath until measured. Data was acquired in BD FACSAria II cytometry.
10,000 events were record for analysis. The first sample would be re-measured at the end of measurement to confirm the stability of signal.
Cyto-ID Autophagy Detection Kit (Enzo Life Science) was used to measure autophagic vacuoles. We performed that according to the instruction manual from manufacturer. To avoid interference, we used HeLa cells that did not expressed GFP-LC3.
Magic Red Cathepsin B Assay Kit (ImunoChemistry) was engaged to evaluate the activity of cathepsin B, a lysosomal cysteine protease. We performed it on HeLa cells that did not expressed GFP-LC3, according to the manufacture's guide.

Methyl thiazolyl tetrazolium (MTT) assay
Cells were seed in 96-well plate at a density of 3 x 10 3 /well (for two days) or 5 x 10 3 /well (for one day). 12 h or 24 h later, replaced by the fresh medium containing nanoparticles. We set six parallel wells each group. After 24 h or 48 h, five of the parallel wells were refreshed by medium containing 0.5 mg/mL MTT. To eliminate the absorption of cells and nanoparticles, we did not add MTT into the sixth well. 4 h later, discarded the medium and replaced with 100 μL dimethylsulfoxide (DMSO).
Shook for 5 min and measured absorption at 490 nm or 570 nm on microplate reader (Multiskan FC, Thermo Scientific). Note: In most cases, there is no difference between measuring at 490 nm and 570 nm.