1. Volume 24, Issue 10, Pages 1783-1796 (October 2016)
Edible Ginger-derived Nano-lipids Loaded with Doxorubicin as a Novel Drug-delivery Approach for Colon Cancer Therapy 
Mingzhen Zhang, Bo Xiao, Huan Wang, Moon Kwon Han, Zhan Zhang, Emilie Viennois, Changlong Xu, Didier Merlin 
Molecular Therapy 
Volume 24, Issue 10, Pages (October 2016)
DOI: /mt
Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

2. Figure 1
Preparation of DOX-FA-GDNVs nanovectors from ginger derived lipids and schematic diagram of the targeted antitumor effect in vivo. (a) Ginger derived nanoparticles (NPs) were isolated and purified from edible ginger by ultracentrifugation (150, 000×g) and sucrose density gradient (8%/30%/45%/60%), the lipids isolated from ginger derived nanoparticles were reassembled in GDNVs, simultaneously, GDNVs could be modified with targeting ligand folic acid mediating targeted delivery of chemotherapy drug (Dox). (b) The schematic diagram showed that i.v. injection of DOX-FA-GDNVs could achieve targeted delivery of chemotherapy drug to tumors through blood vessel. GDNVs, ginger-derived nanovectors; DOX, doxorubicin; FA, folic acid.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

3. Figure 2
Characterization of ginger-derived nanovectors (GDNVs) prepared from ginger-derived lipid. (a) Ginger juice was purified by sucrose density gradient (8%/30%/45%/60%) under ultracentrifugation, band from the interface of 30%/45% (as marked in red rectangle) was harvest and note as ginger derived nanoparticles (NPs) according to literature for further use. (b) Ginger derived NPs harvest from sucrose density gradient (30%/45%) were characterized by transmission electron microscopy (TEM), the scale bar indicates 100 nm. (c) Ginger derived NPs harvest from sucrose density gradient (30%/45%) were also characterized by atomic force microscopy (AFM), the scale bar indicates 1 μm. (d) Pie chart of lipid profile of ginger derivered NPs was indicated in the percentage of total lipids. The lipid composition of ginger derivered NPs was determined by using a triple quadrupole mass spectrometer. DGDG, digalactosyldiacylglycerol; MGDG, monogalactosyldiacylglycerol; LPG, lysophosphatidylglycerol; LPC, lysophosphatidylcholine; LPE, lysophosphatidylethanolamine; PC, phosphatidylcholine; PG, phosphatidylglycerol; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine; PA, phosphatidic acid, (n = 5). (e) GDNVs were purified from the sucrose gradient as marked in red rectangle. (f) Purified GDNVs were characterized by TEM, the scale bar indicates 500 nm. (g) High-resolution TEM image of GDNVs, the scale bar indicates 100 nm. (h) Particle size of GDNVs were measured by measured by dynamic light scattering (DLS) using a Zetesizer Nano ZS. (i,j) Purified GDNVs were characterized by AFM, the scale bar indicates 1 μm.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

4. Figure 3
Evaluation the uptaken efficiency of ginger-derived nanovectors (GDNVs) by Colon-26 and HT-29 cancer cells and potential endocytosis pathway. (a) Confocal images of Dil-labeled GDNVs taken up by Colon-26 cells and HT-29 cells. Cells were incubated with DiL-labeled GDNVs for 4 hours (red channel) and then labeled with phalloidin-FITC (green channel) and 4,6-diamidino-2-2-phenylindole (DAPI) (blue channel) to display the distribution of F-actin and nucleus. The scale bar indicates 20 μm. (b) Quantitative flow cytometry analysis of DiL-labeled GDNVs taken up by Colon-26 cells and HT-29 cells. (c) Potential endocytosis pathway utilized by GDNVs to enter Colon-26 cells. Endocytosis inhibitors (amiloride 34 mg/ml, indomethacin 18 mg/ml, chlorpromazine 4.5 mg/ml, and cytochalasin D 2.5 mg/ml) were first coincubated with Colon-26 cells for 1 hour, and then DiL labeled GDNVs nanovectors were added for 4 hours incubation at 37ºC. Followed, cells were fixed for fluorescence imaging. Scale bar indicates 20 μm. (d) Uptaken efficiency was quantified by flow cytometry (n = 5).
Molecular Therapy  , DOI: ( /mt )
Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

5. Figure 4
Ginger-derived nanovectors (GDNVs) can be used as a drug carrier and load therapeutic agent-doxorubicin (Dox) efficiency. (a) Loading efficiency of Dox in GDNVs was measured. 200 μg of Dox solution was added into the ginger lipid film (the lipid concentrations of 5, 10, 20, 50, 100, and 200 μmol/l were tested), Dox-GDNVs nanovector were centrifuged at 100,000×g for 30 minutes. The supernatant was collected and the residual Dox was measured using the microplate reader at the wavelength of 497 nm. (b) Morphology of Dox-GDNVs was observed by TEM. (c) Particles size of Dox-GDNVs was measured by dynamic light scattering (DLS) using a Zetesizer Nano ZS. (d) Zeta potential of Dox-GDNVs was measured by Zetesizer Nano ZS. (e) The stability of Dox-GDNVs was evaluated. Dox-GDNVs were suspended in buffer (pH 7.4) and stored for 25 days at 4ºC. The stability of Dox-GDNVs was indicated by size and zete potential change. Data are Mean ± SEM. of three independent experiments.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

6. Figure 5
Evaluation the apoptosis in Colon-26 cells induced by free doxorubicin (Dox) and Dox-GDNVs using Annexin V-FITC/PI staining and ECIS technology. (a) Control group. (b) Free Dox group. (c) Dox-GDNVs group. Cells were treated with three different Dox concentrations (3.25, 6.5, and 13 μmol/l) for 8 hours, and the apoptosis were evaluated by FACS using annexin V-FITC/PI staining. (d) Cell viability in each concentration were quantified and compared (n = 3, **P < 0.01, ***P < 0.001). (e) Apoptosis of Caco2-BBE cells manolayers induced by Dox-GDNVs and free Dox was evaluated by real-time ECIS technology. GDNVs, ginger-derived nanovectors; FITC, fluorescein isothiocyanate; PI, propidium iodide; ECIS, electric cell-substrate impedance-sensing.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

7. Figure 6
Anticancer effect of Dox-FA-GDNVs was evaluated in vivo using Colon-26 xenograft mouse model. (a) Stability of circulating GDNVs by observing the fluorescence of DiR. DiR-labeled GDNVs were i.v. injected into mice and the whole blood from mice was collected at different time points (1, 3, 6, 24, and 48 hours) after i.v. injection. The DiR signals from whole blood, blood cells and blood plasma were measured and imaged. The images are representative of three times independent experiments (n = 3). (b) Treatment protocol used in this study. Female athymic BLAB/c nu/nu mice were implanted with Colon-26 cells subcutaneously in the right flank at day 0, from day 8, mice at each group were treated every 4 days for 3 times. At the end of the experiments, mice were sacrificed and the anticancer effects in each group were evaluated and compared (n = 5). (c) Tumor growth profiles in different treatment groups (saline, free Dox, FA-GDNVs and Dox-FA-GDNVs). Arrows indicate the injection time (8, 12, and 16 days after i.v. injection); each point represents the mean ± SEM (n = 5). (d) Body weight changes in different treatment groups. Mouse body weight was normalized to that at the time of injection; each point represents the mean ± SEM (n = 5). (e) Xenografts from each group were imaged and compared at the end of the experiments (n = 5). (f) Tumor volumes at the end of the experiments were compared (n = 5). (g) Tumor weights at the end of the experiment were compared (n = 5). (h) Tumor tissues were removed, fixed and sectioned. H&E-staining of tumor tissues from each group were used to evaluate the antitumor effects. Scale bar: 50 μm. *P < 0.05, **P < GDNVs, ginger-derived nanovectors; Dox, doxorubicin; H&E, hematoxylin and eosin; FA, folic acid.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

8. Figure 7
Effect of inhibiting cell proliferation and stimulation of apoptosis by Dox in Colon-26 xenografts was enhanced by Dox-FA-GDNVs. (a) Immunohistochemical analysis of Ki67 and TUNEL staining of apoptosis cells in Colon-26 xenograft tissues. (b) Rate of cell proliferation in Colon-26 xenograft tissues from different treatment groups. (c) Frequency of apoptosis in Colon-26 xenograft tissues from different treatment groups. Results are presented as mean ± SEM (n = 5). ***P < Scale bar: 50 μm. GDNVs, ginger-derived nanovectors; Dox, doxorubicin; FA, folic acid; SEM, standard error of mean; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end labeling.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

9. Figure 8
Histological analysis were performed to evaluate toxicity of Dox-FA-GDNVs. H&E-stained sections of major organs obtained from tumor-bearing mice treated with saline, free Dox, FA-GDNVs or Dox-FA-GDNVs obtained at the end of the experiment. Scale bar: 50 μm. GDNVs, ginger-derived nanovectors; Dox, doxorubicin; FA, folic acid; H&E, hematoxylin and eosin.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions