(B) The complexation and condensation of CS-gp90. in an osmotic phosphate-buffered saline (PBS) ice bath and collected by centrifugation. After 3 washes, the erythrocyte membranes were resuspended in PBS and mixed with CS-gp90 for 18?h at 4C (18). The solutions were combined and passed 20 times through a liposome extruder with a 200-nm pore diameter filter membrane, and then RAB7B centrifuged to remove excess erythrocyte membranes. After stirring for 1?h, 0.1 mg/mL of DSPE-PEG-MAN solution was added to insert mannose into the erythrocyte membranes to yield mannose-embedded chicken erythrocyte membranes. 3.3. Physicochemical properties of CS-gp90@M-M nanoparticles Agarose gel electrophoresis was used to detect whether the gene was correctly inserted into the plasmid. The ultra-microstructure of the erythrocyte membraneCencapsulated nanoparticles (CS-gp90@M-M) was observed using transmission electron microscopy (TEM) (FEI, Tecnai G2 F20 S-TWIN, CA, USA) and the images were photographed. DSPE-PEG-Man, CS-gp90@M, and CS-gp90@M-M Saquinavir spectra were obtained using Fourier-transform infrared (FT-IR) spectroscopy (8400S, Shimadzu Co., Nakagyo-ku, Kyoto, Japan). IR spectra were recorded and analyzed in the range of 400 to 4000 cm?1. CS-gp90@M and CS-gp90@M-M samples were mounted on glass slides and analyzed using Raman spectroscopy (Horiba, LabRAM, France) at 532 nm in the range of 1000 C 2000 cm?1. The particle size and polymer dispersity index (PDI) of the nanoparticles were determined using a laser particle-size analyzer. Various nanoparticles were simultaneously placed in a potential cup to measure their zeta potential. A standard curve of a CS-gp90 solution was constructed, and the absorbance of the samples was measured at 470 nm. The CS-gp90 concentration in the supernatant was determined using the standard curve. The adsorption rate and drug-loading rate were calculated using formulas (1) and (2) (19). experiments 3.4.1. Cytotoxicity of CS-gp90@M-M nanoparticles cytotoxicity tests were performed using the MTT method (20). Briefly, Saquinavir 2 104/mL HD11 cells were added to 96-well cell culture plates and treated with various concentrations of CS-gp90@M and CS-gp90@M-M nanoparticles at 40 C. After 24?h, 100 L of medium and 10 L of MTT (5 mg/mL) were added for 4?h and the supernatant was aspirated after a purple precipitate was formed. Next, 150 L of dimethyl sulfoxide was added to each well. The absorbance of each well was determined using a microplate Saquinavir reader (Bio-Rad, iMark, Hercules, CA, USA) at 570 nm. 3.4.2. Uptake of CS-gp90@M-M nanoparticles by HD11 cells Cell uptake assay. HD11 cell solution (1 mL) at a concentration of 8 104 cells/mL was added to a 24-well plate, and 50 L of DiD-stained CS-gp90@M and CS-gp90@M-M (200 g/mL) nanoparticles were filtered. After incubation for 8?h in the dark, the culture medium was discarded, and the cells were washed twice with PBS, stained with DAPI dye, and washed twice for 20?min. A drop of anti-fluorescence quenching agent was added to the cells on a clean slide. The slide was analyzed using confocal laser scanning microscopy (CLSM) (ZEISS, Oberkochen, Germany). Quantitative test to determine cell uptake activity. DiD-stained CS-gp90@M (50 L) and CS-gp90@M-M (200 g/mL) nanoparticles were incubated Saquinavir with 5 105 HD11 cells in a Petri dish for 8?h. The intracellular fluorescence intensity of DiD was measured using a small animal imager (Perkin Elmer IVIS LuminaIII, Waltham, MA, USA). 3.4.3. NO secretion and iNOS mRNA expression Cells in the Saquinavir logarithmic growth phase were seeded in a 24-well cell culture plate at a density of 1106 cells/mL. Based on the concentration of the plasmid, 100 g/mL of the nanoparticle solutions prepared using 1 mL of DMEM basic culture medium (PBS group, gp90 plasmid group, CS-gp90 nanoparticle group, CS-gp90@M nanoparticle group, CS-gp90@M-M nanoparticle group) and the positive lipopolysaccharide (LPS) control (1 g/mL) were cultured for 24?h. The nitric oxide (NO) content was determined using an NO detection kit (Nanjing, China). The absorbance was determined using a.