Supporting information Influence of Surface Coatings of PLGA Particles on HepG2 Cell Behavior and Particle Fate Dahai Yu 1, Yuying Zhang 1, Guangyang Zou, Xiaojing Cui, Zhengwei Mao 1* , Changyou Gao 1,2* 1 MOE Key Laboratory of Macromolecular Synthesis and Functionalization, Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, China 2 State Key Laboratory of Diagnosis and Treatment for Infectious Diseases, First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003, China *Corresponding author. Email: email@example.com, firstname.lastname@example.org Fax: +86-571-87951108 100 PLGA-PEI PLGA-BSA Mass(g) 80 60 40 20 0 0 6 Time (h) 10 20 Figure S1. Protein mass of the cells after treated with particles as a function of incubation time. After the cells were incubated with 50 g/mL PLGA particles for 24 h (which is designated as the 0 time point in Figure S1), the total protein content of the cells was measured via a BCA kit as a function of culture time (the exocytosis time). Figure S1 shows that the cells did not duplicate during the exocytosis time until 20h since their protein content kept unchanged. Therefore, exocytosis rather than cell proliferation was the major reason for the decrease of fluorescent intensity of each cell during this period. * PLGA-PEI PLGA-BSA 600 * 400 * * 200 * 0 (A) 0 50 100 Concentration (μg/ml) Average fluorescence intensity per cell Average fluorescence intensity per cell 800 PLGA-PEI PLGA-BSA 600 * * * 400 * * 200 150 (B) 0 0 5 10 15 20 25 Time (h) Figure S2. Uptake of the PLGA-PEI and PLGA-BSA particles by HepG2 cells as a function of (A) particle concentration with a culture time of 3 h, and (B) culture time with a particle concentration of 50 µg/mL. Data were measured by flow cytometry and averaged to each cell. Asterisk indicates significant difference at p < 0.05 level vs respective inhibitors free control. Figure 2 As shown in Figure S2A, the PLGA particles that internalized into or adsorbed onto the cells increased almost linearly along with the increase of particle concentration regardless of their surface chemistry at a fixed co-culture time of 3 h, but the PLGA-PEI particles were more significantly uptaken than the PLGA-BSA particles at each fixed particles concentration (p<0.05). When the particles concentration was fixed at 50 µg/mL, the cellular endocytosis amount of both types of PLGA particles increased significantly along with the co-culture time, especially during the first 6 h (Figure S2B), suggesting the fast cellular endocytosis rate. Again, the PLGA-PEI particles were uptaken with a larger amount at each detection time interval (p<0.05). This result is apparently caused by the difference in surface chemistry. Cell mortality rate (%) 30 Control PLGA-PEI PLGA-BSA 20 10 0 Control 10 25 50 100 Concentration (g/mL) 150 Figure S3. Percentage of dead cells as a function of particle concentration with a culture time of 24 h. The particle free cells were used as a control (about 6% dead cells). The ratios of dead cells after exposure of the PLGA particles of various concentrations were determined by PI staining. As shown in Figure S3, about 90% of the cells survived even after 24 h co-incubation with the PLGA particles of the highest concentration (150 µg/mL). The results suggested that both types of PLGA particles did not cause significant impact on cell mortality. Table S1. Influence of PLGA particles on HepG2 cell cycle. G1 (%) S (%) G2/M (%) Particle free control 54.4±1.3 18.4±1.3 27.1±2.6 PLGA-PEI (50μg mL−1） 54.4±1.7 18.1±1.1 27.6±0.8 PLGA-PEI (150μg mL−1） 51.4±0.2 17.8±0.6 30.8±0.7 PLGA-BSA (50μg mL−1） 57.7±2.0 15.9±0.8 26.5±1.2 PLGA-BSA (150μg mL−1） 57.4±0.8 16.8±1.1 25.8±1.4 Cell cycle is a series of events which can induce cell division and duplication, comprising interphase gap1 (G1), synthesis (S), gap2 (G2) and mitosis (M) phases. Therefore, the cell cycle analysis is powerful to reflect the influence of cellular endocytosis on cell proliferation. Several studies have been focused on the influence of the particles, genes and inhibitors on the cell cycle.[1-4] Since G2 and M phases could not be distinguished only by DNA content, they were put together (G2/M) to represent cell division period. As shown in Table S1, no significant difference of the cell cycle for all the samples was found, revealing that exposure to both types of PLGA particles did not cause obvious influence on cell cycle. Relative cell adhesion (% ) 150 120 90 60 30 (A) 0 PLGA-PEI PLGA-BSA A) 25μm (B) B) (C) (D) C) Figure S4. (A) Cell adhesion percentage after the cells were pre-cultured without or with 50 μg/mL PLGA-PEI and PLGA-BSA particles for 24 h, respectively. Cytoskeleton organization of HepG2 cells treated without (B) or with (C) 50 μg/mL PLGA-PEI and (D) PLGA-BSA particles, respectively. The F-actin was stained by rhodamine-conjugated phalloidin. Figure S4A shows that the adhesion ability of the cells did not change significantly after exposure to both types of the PLGA particles. F-actin staining was performed to examine the effects of the PLGA particles on the cytoskeleton of HepG2 cells. As shown in Figure S4B, the microfilaments in the control cells were well ordered. After the cells were exposed to 50 µg/mL PLGA particles for 12 h, the organization of microfilaments changed to some extent (Figure S4C, D), especially of the cells treated with PLGA-PEI particles (Figure S4C). (A) 20 -20 -15 -10 -5 15 15 10 10 5 5 0 0 -5 0 5 10 15 20 25 -25 -20 -15 -10 -5 -5 (C) 20 (B) 20 B -25 25 25 25 15 10 5 0 0 5 10 15 20 25 -25 -20 -15 -10 -5 -5 0 5 10 15 20 25 A A -10 -10 -10 -15 -15 -15 -20 -20 -20 -25 -25 -25 Migration Rate (μm/h) 10 8 * 6 * 4 2 (D) 0 Control PLGA-PEI PLGA-BSA Figure S5. Migration trajectories of HepG2 cells pre-cultured without (A) or with 50 µg/mL (B) PLGA-PEI and (C) PLGA-BSA particles. Quantitative analysis of the cell migration rate (D). Asterisk indicates significant difference at p < 0.05 level. The cell migration plays a central role in a wide variety of biological phenomena. It is a dynamic adhesion process requiring coordinate activity of cytoskeleton, membrane and signaling system. The cell mobility was reduced to some extent after the cells were exposed to 50 µg/mL PLGA-PEI particles for 24h (p<0.05). 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