Photo-derived transformation from modified chitosan@calcium carbonate nanohybrids to nanosponges

Zwitterionic chitosan (ZC)@calcium carbonate (CC) nanoparticles were conveniently obtained and transformed to biocompatible nanosponges by continuous gas-phase photo-derived transformation in a single-pass configuration, and their potential use for biomedical applications was investigated. The mean diameter of the ZC@CC sponges was ~166 nm (~72 nm for CC and, ~171 nm for ZC), and the sponges had a mesoporous structure (i.e., an average pore diameter of ~13 nm). Measurements of the sponge cytotoxicity were performed and only a slight decrease was observed (>78% in cell viability) when compared with pure ZC (>80%). The ZC@CC sponges had a similar transfection ability to lipofectamine (~2.7 × 109 RLU mg−1 protein) at a 50:1 ratio of sponge:DNA weight. Because of a porous structure, the sponges showed remarkably higher transfection efficiencies than pure ZC.


-Instrumentation
The size distributions of the fabricated 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 was used to measure 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 carbon dioxide 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 gas-phase 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).
Scanning electron microscope (SEM, NOVA nanoSEM, FEI, US) images for the CC-ZC particles were obtained at an accelerating voltage of 15 kV. The nitrogen adsorption isotherms of the ZC@CC sponges were measured using a porosimeter (ASAP 2010, Micromeritics Ins. Corp., US) at 77.4 K at a relative pressure ranging from 10 -6 to 1.
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 sponge/plasmid DNA (pDNA) complexes was determined using a zeta potential analyzer (Nano ZS-90, Malvern Instruments, UK). The particles were mixed with pDNA, and incubated at room temperature for 30 min. The complexes were then diluted with double de-ionized water to an appropriate concentration. Measurements of the zeta potential were carried out at 25 o C and calculated using the manufacturer's supplied software. -

Agarose Gel Retardation Assay
The gene condensation ability of the nanosponges under different weight ratios were analyzed by 1% agarose gel electrophoresis using tris-acetate-ethylenediaminetetraacetic acid buffer (242 g Tris, 57.1 mL glacial acetic acid, and 0.5 mM ethylenediaminetetraacetic acid, pH 8.0) containing 0.5 µg mL -1 ethidium bromide. Complexes containing nanosponges and genes with different weight ratios were prepared by mixing, voltexing, and incubating them at room temperature for 30 min. Approximately 100 ng of each complex was loaded on agarose gels. A gel loading dye blue (New England BioLabs, USA) was added to each well and agarose gel electrophoresis was carried out at a constant voltage of 80 V for 50 min. The gene bands of the resultant gels were then visualized under a ultraviolet transilluminator at a wavelength of 365 nm.

-Macrophage Inflammatory Protein (MIP) 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 Janus particle solution was injected to each well to set the particle concentraion in medium to 2 mg mL -1 . For comparison purposes, 0.1 mL of polyethyleneimine (PEI, 765090, Sigma-Aldrich, USA), poly-l-lysine (PLL, P4707, Sigma-Aldrich, USA), or polyethylene glycol (PEG, 81188, Sigma-Aldrich, USA) was injected in lieu of the ZC precursor solutions. After 24 h incubation, the culture media were centrifuged at 2000 rpm for 10 min to separate supernatants. Macrophages were challenged by adding lipopolysaccharide (LPS) to the media in the final concentration of 1 μg mL -1 shortly before the comparisons. 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. Figure S1. TEM images for the other chitosan@CC nanohybrids.

-TEM images for the other chitosan@CC nanohybrids
The TEM images (Fig. S1) indicate that the morphology of the CC particles is an elliptical shape, while pure chitosan (Cs) and cholesterol-chitosan (Ch-Cs) particles exhibit a similar spherical shape with a smooth surface and are separate from each other. When the CC particles passed through the orifice of the atomizer, the CC particles were capsulated by Cs or Ch-Cs particle due to the gas pressurizing system. The TEM images show the gray outer shell around CC nanoparticles, implying the presence of a Cs or Ch-Cs moiety that completely covers the CC particles. However, there was no porous chitosan network on the CC particles, dissimilar to the ZC@CC configuration, although they were fabricated by the same method used to fabricate the ZC@CC. This difference may have originated from the differences in the UV sensitivity and solubility in water among the configurations.