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Cytokine Assays

After conjugation, PLGA and Chitosan nanoparticles were precipitated and the free amounts of rHCA59 protein were determined by a BCA protein assay kit. It was found that 89% and 71% of rHCA59 were conjugated to PLGA and Chitosan, respectively. In other words, a total of 1.78 mg of protein bound to PLGA, and 1.42 mg of protein bound to Chitosan.A scanning electron microscopy was used to identify the size and morphology of PLGA and Chitosan nanoparticles. The SEM results showed that both PLGA and Chitosan nanoparticles’ appearances were smooth. The size of rHCA59-PLGA NPs was from 180 nm to 402 nm (Figure 1A) and the size of rHCA59-CS NPs was from 200 nm to 500 nm (Figure 1B).

Notes: (1): PBS group (blank control). (2): pET-32a protein group. (3): PBS-PLGA group. (4): PBS-CS group. (5): PBS-CFA group. (6): rHCA59 group. (7): rHCA59-PLGA group. (8): rHCA59-CS group. (9): rHCA59-CFA group. (10): rHCA59-CS-PLGA group. (A) IL-12. (B) IL-17. (C) IFN-γ. (D) IL-4. (E) TGF-β. Data are representative of triplicate independent experiments (*p < 0.05, **p < 0.01, and ***p < 0.001). Abbreviations: PLGA, poly-D,L-lactide-co-glycolide; CS, Chitosan; CFA, complete Freund’s adjuvant; IL, interleukin; ELISA, enzyme linked immunosorbent assay

Notes: rHCA59 loaded nanoparticles could affect the proportions of CD4+ T cells and CD8+ T cells in immunized mice. The percentages of CD4+ T cells (A1–A10) and CD8+ T cells (B1–B10) in ten groups were investigated by flow cytometry on day 14 after immunization. (C and D) Different treatments affected the proportions of CD4+ T cells and CD8+ T cells. (A1 and B1) PBS group (blank control). (A2 and B2) PBS-CFA group. (A3 and B3) PBS-PLGA group. (A4 and B4) PBS-CS group. (A5 and B5) pET-32a protein group. (A6 and B6) rHCA59 group. (A7 and B7) rHCA59-CFA group. (A8 and B8) rHCA59-CS group. (A9 and B9) rHCA59-PLGA group. (A10 and B10) rHCA59-CS-PLGA group. The results shown here are from three independent experiments (*p < 0.05, **p < 0.01, and ***p < 0.001). Abbreviations: CD4, cluster of differentiation 4; CD8, cluster of differentiation 8; T cells, thymus cells; PLGA, poly-D,L-lactide-co-glycolide; CS, Chitosan; CFA, complete Freund’s adjuvant.