Easy on-demand single-pass self-assembly and modification to fabricate gold@graphene-based anti-inflammatory nanoplatforms

Zwitterionic chitosan (ZC) was modified by fully (both for lateral dimension and thickness) nanodimensional gold-graphene oxide (Au@GO) flakes under visible light and the potential of the resulting materials as biomedical nanoplatforms was investigated. Fully nanodimensional GO flakes floating in nitrogen gas were incorporated with Au nanoparticles to form Au@GO nanoflakes, and the Au@GO was then incorporated with ZC droplets to form the Au@GO-ZC hybrid nanoparticles. The collected particles were exposed to visible light to induce the photocatalytic activity of the Au@GO nanoflakes towards the ZC derivatives. The visible-light-exposed particles show different chemical and surface properties from the unexposed particles, while there were no significant differences in cytotoxicity and macrophage inflammatory protein production. This work suggests that incorporating fully nanodimensional Au@GO flakes with ZC is a suitable technique for ambient photo-modification of the chitosans’ surface property without significant changes in size and shape and increases in cytotoxicity and inflammatory response.


-Fabrication
Graphite was obtained from a spark discharge system. S1 GO was prepared from the graphite by using a modified Hummer's method. S2 Schematic diagrams of the aerosol-based method used for these experiments are shown in Fig. 1 Freshly spark generated Au was first passed over a collison atomizer containing GO to form AuGO nanoparticles. A pure nitrogen (99.999% purity) flow, which was controlled by a mass flow controller (3810DS, Kofloc, Japan), had a flow rate of 3 L min -1 to carry particles. After passed through a diffusion dryer, the AuGO-laden flow was directly employed to further atomize the ZC precursor solution. The ZC precursor solution was used from a method of Xu et al. S3 The AuGO-ZC hybrid droplets then passed through a heated tubular reactor with a 90 o C wall temperature for the solvent extraction of the droplets. The condition for complete evaporation can be estimated by considering the time required for the evaporation of the droplets and comparing it with the appropriate residence time in the tubular reactor. The characteristic time to saturate gas with vapor from evaporating droplets, τ, is given via the equation, where D d is the diameter of the droplet, δ v is the diffusivity of the vapor, and C(D d ) is the droplet number concentration.

-Instrumentation
The size distributions of the aerosol particles were measured using a scanning mobility particle sizer (SMPS), consisting of a differential mobility analyzer (3081, TSI, US), electrostatic classifier (3080, TSI, US), condensation particle counter (3776, TSI, US), and a soft X-ray charger (4530, HCT, Korea). The SMPS system, which measured the mobility equivalent diameter, was operated at a sample flow of 0.3 L min -1 , a sheath flow of 3.0 L min -1 , and a scan time of 135 sec (measurement range: 15.1-661.2 nm). The mass (m) of the fabricated particles was measured using a microbalance (DV215CD, Ohaus, Switzerland) and also confirmed via the following equation: where Q is the flow rate of nitrogen gas, t s is the sampling time, η(D p ) is the fractional collection efficiency, and C m (D p ) is the mass concentration of particles.
Transmission electron microscope (TEM, CM-100, FEI/Philips, US) images were obtained at an accelerating voltage range of 46-180 kV. Specimens were prepared for examination in the TEM by direct electrostatic aerosol sampling at a sampling flow of 1.0 L min -1 and an operating voltage of 5 kV using a nano particle collector (NPC-10, HCT, Korea).
For Fourier transform infrared (FTIR) spectroscopy analysis, samples were prepared using polytetrafluoroethylene (PTFE) media substrate (0.2 μm pore size, 47 mm diameter, 11807-47-N, Sartorius, Germany) by physical filtration (i.e. mechanical filtration mainly by diffusion, of particles on the surfaces of the substrate), and the spectra were recorded on a Nicolet 6700 FTIR spectrometer (Thermo Electron, US). The spectra were taken for samples in the range of 4000-400 cm -1 in absorbance mode.
The zeta potential of the fabricated particles was determined using a zeta potential analyzer (Nano ZS-90, Malvern Instruments, UK). Measurements of the zeta potential were carried out at 25 o C and calculated using the manufacturer's supplied software.

-In Vitro Cytotoxicity
The cytotoxicity of the samples was evaluated using HeLa cells by MTS, 3-(4,5-dimethyl-thiazol-2-yl)- performed using Student's t-test. The differences were considered significant for p < 0.05.

-Macrophage Inflammatory Protein Production
Peritoneal macrophages were seeded in 24-well plates at a density of 10 5 cells per well in 1 mL of medium. After overnight incubation, 0.1 mL of the sample particle solution was injected to each well to set the particle concentraion in medium to 2 mg mL -1 . In control groups, 0.1 mL of PBS or unmodified chiotosan was injected in lieu of the sample particle solutions. After 24 h incubation, the culture media were centrifuged at 2000 rpm for 10 min to separate supernatants. Macrophages were challenged by adding LPS to the media in the final concentration of 1 μg mL -1 shortly before the Cs and PBS controls.
For sample particles, enzyme-linked immunosorbent assay (ELISA) was performed to determine the MIP levels using MIP-2 ELISA kit (R&D Systems, USA). The supernatants collected from LPS-challenged macrophages was always diluted 10 times prior to the analysis. The differences were considered significant for p < 0.01.