Figures & data
Figure 1 TEM (top panel) and SEM (bottom panel) images of DOX/FA-Z-NCs (A, C) and DOX/Z-NCs (B, D).
Note: The inset in (A) shows a typical example of the hollow nanocapsule (FA-Z-NC) in the magnified view.
Abbreviations: DOX, doxorubicin; FA, folic acid; NCs, nanocapsules; SEM, scanning electron microscopy; TEM, transmission electron microscopy.
Figure 2 DOX release from nanocapsules triggered by various reduction conditions.
Notes: (A) DOX/FA-Z-NCs; (B) DOX/Z-NCs.
Abbreviations: DOX, doxorubicin; FA, folic acid; GSH, glutathione; NCs, nanocapsules; PBS, phosphate buffer saline.
Figure 3 In vitro cytotoxicity assay.
Note: 4T1 cells were incubated with DOX-containing formulations for 36 h.
Abbreviations: DOX, doxorubicin; DOX·HCl, doxorubicin hydrochloride; FA, folic acid; NCs, nanocapsules.
Figure 4 Flow cytometry results of time-dependent DOX signal changes.
Note: (A) 2 h and (B) 24 h.
Abbreviations: DOX, doxorubicin; DOX·HCl, doxorubicin hydrochloride; FL2-A, fluorescent signal of DOX; FA, folic acid; NCs, nanocapsules.
Figure 5 CLSM images of 4T1 cell lines incubated with DOX·HCl, DOX/FA-Z-NCs, and DOX/Z-NCs for 0.5, 2, and 6 h.
Notes: The fluorescent channels show signals of DOX (red), lysotracker green-labeled acidic endolysosomes (green), and the merged images (orange/yellow). Scale bar measures 20 µm.
Abbreviations: CLSM, confocal laser scanning microscopy; DOX, doxorubicin; DOX·HCl, doxorubicin hydrochloride; FA, folic acid; NCs, nanocapsules.
Figure 6 In vivo antitumor efficacy.
Notes: Variations in (A) tumor volume and (B) body weight of the tumor-bearing BALB/c mice (n=5). In (A), the DOX/FA-Z-NCs demonstrated significant tumor inhibition effect (**p<0.001, compared to saline; *p<0.05, compared to DOX·HCl). In (B), the saline and DOX/NCs groups showed no significant effect on body weight, whereas the DOX·HCl induced obvious body weight loss (*p<0.05, compared to DOX·HCl). The arrows represent all the five injections during the experimental period.
Abbreviations: DOX, doxorubicin; DOX·HCl, doxorubicin hydrochloride; FA, folic acid; NCs, nanocapsules.
Figure 7 The (A) ex vivo fluorescence images and (B) average signals of excised major organs and tumor tissue at different time intervals (2, 7, and 24 h) postadministration in (B) DOX·HCl, (C) DOX/Z-NCs and (D) DOX/FA-Z-NCs groups.
Notes: The fluorescent signals were collected based on emission of the DOX, and the dosage of the injected DOX was normalized to be 5 mg/kg. The sample groups were placed in the order DOX·HCl, DOX/Z-NCs, and DOX/FA-Z-NCs in each panel. Average signals were collected by using the Maestro in vivo imaging system.
Abbreviations: DOX, doxorubicin; DOX·HCl, doxorubicin hydrochloride; FA, folic acid; NCs, nanocapsules.
Figure 8 Immunohistochemical staining of tumor tissues.
Notes: (A) Ki 67 positive (brown) and TUNEL positive (brown) represent tumor cell proliferation inhibition and tumor apoptosis, respectively. Quantification of the (B) Ki 67–positive areas and (C) TUNEL-positive areas. In (A), scale bar measures 40 µm. In (B and C), the DOX-containing formulations exhibited significant tumor cell death (*p<0.05, **p<0.001).
Abbreviations: DOX, doxorubicin; DOX·HCl, doxorubicin hydrochloride; FA, folic acid; NCs, nanocapsules; TUNEL, transferase-mediated deoxyuridine triphosphate-biotin nick-end labelling.
Figure 9 HE staining of tissues of major organs (heart, liver, spleen, lung, kidney; ×100).
Notes: For the DOX·HCl group: black arrows point out granular degeneration of the cardiac muscles and the black line marks laminar necrosis of liver cells. For the saline group: the black arrows point out diffuse hepatic necrosis and nucleus dissolution necrosis in the liver and inflammatory cell infiltration in the lung; the blue arrows point out tumor cell metastasis into the lung. Scale bar measures 100 µm.
Abbreviations: DOX, doxorubicin; DOX·HCl, doxorubicin hydrochloride; FA, folic acid; HE, Hematoxylin and Eosin; NCs, nanocapsules.
Scheme 1 Active targeting of the nanocapsules and intracellular drug trafficking.
Abbreviations: DOX, doxorubicin; FA, folic acid; HA, hyaluronic acid; NCs, nanocapsules; O/W, oil in water.
