Figures & data
Figure 1 Synthesis route to Cs-N3, the azido-functionalized chitosan substituted derivative, poly (L-lysine) G3 dendron-conjugated chitosan (Cs-g-PLLD), and Cs-g-PLLD-FA.
Abbreviations: DCC, dicyclohexylcarbodiimide; NHS, N-hydroxysuccinimde; DMSO, dimethyl sulfoxide; FA, folic acid; PLLD, poly (L-lysine) dendrons; Cs-g-PLLD-FA, a novel nanoscale polysaccharide derivative prepared by click conjugation of azido-modified chitosan with propargyl focal point PLLD and subsequent coupling with FA.
Figure 2 Properties of Cs-g-PLLD/siAEG-1 and Cs-g-PLLD-FA/siAEG-1 complexes at various N/P ratios: (A) average particle size and (B) zeta potential after 30 minutes, (C) average particle size and (D) zeta potential after 24 hours. TEM image of the (E) Cs-g-PLLD/siAEG-1 complex and (F) Cs-g-PLLD-FA/siAEG-1 complex formed at an N/P ratio of 20.
Abbreviations: N/P, nitrogen/phosphorus; TEM, transmission electron microscopy; FA, folic acid; PLLD, poly (L-lysine) dendrons; AEG-1, astrocyte elevated gene-1; Cs-g-PLLD-FA, a novel nanoscale polysaccharide derivative prepared by click conjugation of azido-modified chitosan with propargyl focal point PLLD and subsequent coupling with FA.
Figure 3 Electrophoretic migration of AEG-1 siRNA, Cs-g-PLLD/siAEG-1 (A), and Cs-g-PLLD-FA/siAEG-1 (B) complexes at various N/P ratios. (C) The cytotoxicities of Cs-g-PLLD or Cs-g-PLLD-FA in 143B cells were evaluated using the MTT assay.
Note: Error bars represent standard deviations calculated from three independent experiments.
Abbreviations: N/P, nitrogen/phosphorus; FA, folic acid; PLLD, poly (L-lysine) dendrons; AEG-1, astrocyte elevated gene-1; Cs-g-PLLD-FA, a novel nanoscale polysaccharide derivative prepared by click conjugation of azido-modified chitosan with propargyl focal point PLLD and subsequent coupling with FA; siSCR, scrambled small interfering RNA; Lip, Lipofectamine 2000.
Figure 4 (A) Fluorescence images under an inverted fluorescence microscope (×400 magnification) and (B) representative histograms of the percentage of fluorescent cells analyzed by flow cytometry for cy3-siRNA fluorescence 24 hours after the final transfection in 143B cells. Scale bar 50 μm.
Abbreviations: N/P, nitrogen/phosphorus; FA, folic acid; PLLD, poly (L-lysine) dendrons; AEG-1, astrocyte elevated gene-1; Cs-g-PLLD-FA, a novel nanoscale polysaccharide derivative prepared by click conjugation of azido-modified chitosan with propargyl focal point PLLD and subsequent coupling with FA; Lip, Lipofectamine 2000.
Figure 5 Confocal laser scanning microscope images of various complexes in 143B cells after incubation for 4 hours.
Notes: Image assignment: blue, nucleus; red, AEG-1 siRNA. The image on the right is an overlay of the two fluorescent colors (×1,000 magnification). Scale bar 50 μm.
Abbreviations: FA, folic acid; PLLD, poly (L-lysine) dendrons; AEG-1, astrocyte elevated gene-1; Cs-g-PLLD-FA, a novel nanoscale polysaccharide derivative prepared by click conjugation of azido-modified chitosan with propargyl focal point PLLD and subsequent coupling with FA; DAPI, 4′,6′diamidino-2-phenylindole.
Figure 6 (A, B) Knockdown of AEG-1 by siRNA in 143B cells by Western blot analysis and qRT-PCR. GAPDH was used as a loading control. (C) Knockdown of AEG-1 inhibits cell proliferation as measured by the MTT assay in 143B cells. (D) Reduced AEG-1 expression inhibits the invasive ability of 143B cells (×400 magnification). (E) Average number of penetrated 143B cells in the invasion assay.
Notes: Error bars represent standard deviations calculated from three independent experiments. *P<0.05 versus control cells for (B, C, E). Scale bar 50 μm.
Abbreviations: FA, folic acid; PLLD, poly (L-lysine) dendrons; AEG-1, astrocyte elevated gene-1; Cs-g-PLLD-FA, a novel nanoscale polysaccharide derivative prepared by click conjugation of azido-modified chitosan with propargyl focal point PLLD and subsequent coupling with FA; Lip, Lipofectamine 2000; siSCR, scrambled small interfering RNA; qRT-PCR, quantitative reverse transcription-polymerase chain reaction.
Figure 7 (A) Photographs of 143B tumors in all groups of excised tumors. (B) Representative CT scan radiographs (upper) and photographs by H&E staining (lower) of the lungs in all groups before mouse sacrifice. Lung metastatic nodules are indicated by arrows. (C) Immunohistochemical staining of AEG-1, Ki-67, MMP-2, and MMP-9 in all groups. Scale bar 100 μm.
Abbreviations: CT, computed tomography; MMP, matrix metalloprotease; FA, folic acid; PLLD, poly (L-lysine) dendrons; AEG-1, astrocyte elevated gene-1; Cs-g-PLLD-FA, a novel nanoscale polysaccharide derivative prepared by click conjugation of azido-modified chitosan with propargyl focal point PLLD and subsequent coupling with FA; siSCR, scrambled small interfering RNA.
Scheme 1 A schematic summary of synthesis and antitumor process of Cs-g-PLLD-FA/siAEG-1.
Notes: (A) Encapsulating siRNA molecules into the Cs-g-PLLD-FA. (B) Self-assembly of Cs-g-PLLD-FA/siAEG-1. (C) In vivo cancer treatment through injecting Cs-g-PLLD-FA/siAEG-1. (D) In vitro gene silencing process initiated by Cs-g-PLLD-FA/siAEG-1.
Abbreviations: FA, folic acid; PLLD, poly (L-lysine) dendrons; AEG-1, astrocyte elevated gene-1; Cs-g-PLLD-FA, a novel nanoscale polysaccharide derivative prepared by click conjugation of azido-modified chitosan with propargyl focal point PLLD and subsequent coupling with FA.
Figure S1 (A) IR spectra of chitosan, Cs-N3, Cs-g-PLLD, and Cs-g-PLLD-FA. (B) 1H NMR spectra of Cs-N3, Cs-g-PLLD, and Cs-g-PLLD-FA (D2O, 25°C). (C) The chemical structure of Cs-g-PLLD-FA.
Abbreviations: IR, infrared; NMR, nuclear magnetic resonance; FA, folic acid; PLLD, poly (L-lysine) dendrons; Cs-g-PLLD-FA, a novel nanoscale polysaccharide derivative prepared by click conjugation of azido-modified chitosan with propargyl focal point PLLD and subsequent coupling with FA.
Figure S2 The cytotoxicities of Cs-g-PLLD or Cs-g-PLLD-FA in U2OS cells were evaluated using the MTT assay.
Note: Error bars represent standard deviations calculated from three independent experiments.
Abbreviations: FA, folic acid; PLLD, poly (L-lysine) dendrons; Cs-g-PLLD-FA, a novel nanoscale polysaccharide derivative prepared by click conjugation of azido-modified chitosan with propargyl focal point PLLD and subsequent coupling with FA; siSCR, scrambled small interfering RNA; Lip, Lipofectamine 2000.
Figure S3 (A) Fluorescence images under an inverted fluorescence microscope (×400 magnification) and (B) representative histograms of the percentage of fluorescent cells analyzed by flow cytometry for cy3-siRNA fluorescence 24 hours after the final transfection in U2OS cells. Scale bar 50 μm.
Abbreviations: N/P, nitrogen/phosphorus; FA, folic acid; PLLD, poly (L-lysine) dendrons; AEG-1, astrocyte elevated gene-1; Cs-g-PLLD-FA, a novel nanoscale polysaccharide derivative prepared by click conjugation of azido-modified chitosan with propargyl focal point PLLD and subsequent coupling with FA; Lip, Lipofectamine 2000.
Figure S4 Confocal laser scanning microscope images of various complexes in U2OS cells after incubation for 4 hours.
Notes: Image assignment: blue, nucleus; red, AEG-1 siRNA. The image on the right is an overlay of the two fluorescent colors (×1,000 magnification). Scale bar 50 μm.
Abbreviations: FA, folic acid; PLLD, poly (L-lysine) dendrons; AEG-1, astrocyte elevated gene-1; Cs-g-PLLD-FA, a novel nanoscale polysaccharide derivative prepared by click conjugation of azido-modified chitosan with propargyl focal point PLLD and subsequent coupling with FA; DAPI, 4′,6′diamidino-2-phenylindole.
Figure S5 (A, B) Knockdown of AEG-1 by siRNA in U2OS cells by Western blot analysis and qRT-PCR. GAPDH was used as a loading control. (C) Knockdown of AEG-1 inhibits cell proliferation as measured by the MTT assay in U2OS cells. (D) Reduced AEG-1 expression inhibits the invasive ability of U2OS cells (×400 magnification). (E) Average number of penetrated U2OS cells in the invasion assay.
Notes: Error bars represent standard deviations calculated from three independent experiments. *P<0.05 versus control cells for (B, C, E). Scale bar 50 μm.
Abbreviations: FA, folic acid; PLLD, poly (L-lysine) dendrons; AEG-1, astrocyte elevated gene-1; Cs-g-PLLD-FA, a novel nanoscale polysaccharide derivative prepared by click conjugation of azido-modified chitosan with propargyl focal point PLLD and subsequent coupling with FA; Lip, Lipofectamine 2000; siSCR, scrambled small interfering RNA; qRT-PCR, quantitative reverse transcription-polymerase chain reaction.
Figure S6 (A, B) The tumor volume and weight in all groups.
Notes: *P<0.05, n=5, versus the group of tumors treated with Cs-g-PLLD/siSCR. **P<0.05, n=5, versus the group of tumors treated with Cs-g-PLLD/siAEG-1.
Abbreviations: FA, folic acid; PLLD, poly (L-lysine) dendrons; AEG-1, astrocyte elevated gene-1; Cs-g-PLLD-FA, a novel nanoscale polysaccharide derivative prepared by click conjugation of azido-modified chitosan with propargyl focal point PLLD and subsequent coupling with FA; Lip, Lipofectamine 2000; siSCR, scrambled small interfering RNA.
Figure S7 (A) Body weight of the mice in all groups. (B) Representative photographs of the heart, liver, spleen, lung, and kidney sections in all groups by H&E staining (×400 magnification). Scale bar 100 μm.
Abbreviations: FA, folic acid; PLLD, poly (L-lysine) dendrons; AEG-1, astrocyte elevated gene-1; Cs-g-PLLD-FA, a novel nanoscale polysaccharide derivative prepared by click conjugation of azido-modified chitosan with propargyl focal point PLLD and subsequent coupling with FA; siSCR, scrambled small interfering RNA.
