## 2 Materials and Methods
### Cellular Uptake Study on PLGA-PEG-PLGAA1004 NPs Using HaCaT Cells  
We conducted cellular uptake tests on PLGA-PEG-PLGAA1004 NPs using HaCaT cells, a human epidermal keratinocyte cell line. Epidermal thickening, one of the symptoms of psoriasis, is caused by the abnormal differentiation of epidermal cells. Immortalized HaCaT cells are widely used as model cells to mimic this condition [13]. For PLGA-PEG-PLGAA1004 NPs and their comparison, PLGA NPs were prepared using a combination of an antisolvent diffusion method with preferential solvation [14; 15]. An amount of 3 mL of NMP in which 4.8 mg of CsA and 75.2 mg of either polymer were dissolved was injected into 20.0 mL of purified water at a flow rate of 10 mL/s to prepare 30 nm particles [16]. To remove excess NMP and unloaded CsA, the prepared nanoparticle suspension was dialyzed for 24 h in a dialysis tube (UC36-32-100, molecular weight cut-off: 14,000, EIDIA Co., Ltd., Tokyo, Japan) [11]. Nanoparticles used in cellular uptake studies are required to contain a fluorescent substance as a tracer for observation and measurement. We prepared PLGA or PLGA-PEG-PLGAA1004 NPs containing comumarin-6 by replacing CsA with coumarin-6 in the method described above [11] and conducted a cellular uptake test using HaCaT cells. We prepared PLGA or PLGA-PEG-PLGAA1004 NPs containing comumarin-6, as a nanoparticle tracer, using the previously reported method [11] and conducted the cellular uptake tests using HaCaT cells. HaCaT cells were seeded on a chambered culture slide at a concentration of 2 \(\times\) 10\({}^{5}\) cells/mL and incubated for 24 h under a 5% CO\({}_{2}\) atmosphere (37 \({}^{\circ}\)C) to allow cells to adhere [17]. The medium was removed, and 180 uL of DMEM and 20 uL of free coumarin-6, comumarin-6-loaded PLGA NPs, or coumarin-6-loaded PLGA-PEG-PLGAA1004 NPs with coumarin-6 concentration adjusted to 0.02 mg/mL were added and incubated for 2 h. After discarding the supernatant, particles adhering to the cell surface were washed with phosphate-buffered saline (PBS) and fixed with 4% (\(w/v\)) paraformaldehyde. Cells washed with TritonTM X (0.1%) were stained and mounted with 20 uL/well DAPI and observed using a confocal laser scanning microscope (CLSM, Leica TCS SP8, Leica Microsystems, Solms, Germany) [18; 19; 20]. To evaluate the uptake of NPs by HaCaT cells, we measured the fluorescence intensity of coumarin-6 using fluorescence-activated cell sorting (FACS, BD FACSCalibur, BD Bioscience, San Jose, CA, USA). HaCaT cells were seeded in a 6-well plate at a concentration of 5 \(\times\) 10\({}^{4}\) cells/mL, and then incubated in a 5% CO\({}_{2}\) atmosphere (37 \({}^{\circ}\)C) for 24 h to allow the cells to adhere. The medium was removed, and 1800 uL of DMEM and 200 uL of free coumarin-6-loaded PLGA NPs, or coumarin-6-loaded PLGA-PEG-PLGA1004 NPs with coumarin-6 concentration adjusted to 0.02 mg/mL were added and incubated for 2 h. Particles adhering to the cell surface were washed with PBS [20], and after deadhesion treatment was performed using a trypsin-EDTA solution, centrifugation was performed at 1300 rpm for 3 min. The supernatant was discarded, and the precipitate was redispersed in 0.5 mL of PBS. In these experiments, PBS was used as a control.