Figure 1.
Design of DOX loading DOX-hyd-PEG-FA NPs as an active targeting drug delivery system.
(1) FA receptor mediated endocytosis; (2) NPs depolymerization in lysosome; (3) DOX escape from the lysosome; (4) DOX diffuse to nucleus.
Figure 2.
Reaction scheme for the synthesis of DOX-ami-PEG-FA conjugate.
(1) NHS, DCC, DMSO; (2) H2N-PEG-COOH, TEA, DMSO; (3) NHS, DCC, CH2Cl2, TEA.
Figure 3.
Reaction scheme for the synthesis of DOX-hyd-PEG-FA conjugate.
(1) NHS, DCC, DMSO; (2) H2N-PEG-COOH, TEA, DMSO; (3) NHS, DCC, BOCNHNH2, DMSO, TEA; (4) TFA, DMSO; (5) DOX, TFA, DMSO.
Figure 4.
1H NMR spectrum of DOX-hyd-PEG-FA (upper panel) and DOX-ami-PEG-FA (lower panel).
Figure 5.
Typical chromatograms of free DOX (upper panel), DOX-hyd-PEG-FA (middle panel) and DOX-ami-PEG-FA (lower panel).
Table 1.
Characterization of nanoparticles. Data represented mean±SD (n = 3).
Figure 6.
Long-term stability of DOX-ami-PEG-FA NPs (panel A) and DOX-hyd-PEG-FA NPs (panel B) in terms of particle size and polydispersity index (PDI) in PBS at room temperature.
n = 3.
Figure 7.
DOX release profiles from the DOX-hyd-PEG-FA NPs (panel A) and DOX-ami-PEG- FA NPs (panel B) in different pH medium.
**P<0.01 vs pH 7.4 medium at 58 h, n = 3.
Figure 8.
Confocal laser scaning microscopy (CLSM) images of KB cells incubated with DOX (panel A), DOX-hyd-PEG-FA NPs (panel B) and DOX-hyd-PEG NPs (panel C) at 37°C for 4 h. DOX concentration was 10 µg/mL.
The pink region shows the localization of DOX (red) in the nucleus (blue). DOX mean fluorescence intensity (MFI) in the nucleus and cytoplasm of the KB cells are shown in panel D. **P<0.01 vs cytoplasma with the same treatment, n = 5.
Figure 9.
Confocal laser scaning microscopy (CLSM) images of A549 cells incubated with DOX (panel A), DOX-hyd-PEG-FA NPs (panel B) and DOX-hyd-PEG NPs (panel C) at 37°C for 4 h.
DOX concentration was 10 µg/mL. The pink region shows the localization of DOX (red) in the nucleus (blue). DOX mean fluorescence intensity (MFI) in the nucleus and cytoplasm of the KB cells are shown in panel D. **P<0.01 vs cytoplasma with the same treatment, n = 5.
Figure 10.
Panel A-Flow cytometry results of KB cells that were incubated with DOX,DOX-hyd-PEG NPs and DOX-hyd-PEG-FA NPs for 30 min and 2 h.
DOX concentration was 10 µg/mL. PA-peak area. *p<0.05 vs same treatment at 30 min; #p<0.05 vs DOX-hyd-PEG NPs at the same time point, n = 5. Panel B-Flow cytometric analysis of KB cells and A549 cells treated with DOX-hyd-PEG-FA NPs when exogenous folate was absent and present in the culture medium. DOX concentration was 10 µg/mL.Incubation time was 2 h. PA-peak area. *p<0.05 vs DOX-hyd-PEG-FA NPs on KB cells, n = 5.
Figure 11.
Subcellular localization of DOX in KB cells delivered by DOX-ami-PEG-FA NPs and DOX-hyd-PEG-FA NPs.
The pink region shows the localization of DOX (red) in the nucleus (blue), the yellow region shows the localization of DOX (red) in the lysosome/endosome (green).
Figure 12.
Effect of exogenous free FA on the viability of KB cells incubated with DOX-hyd-PEG-FA NPs for 48 h. DOX concentration was 0.6 µg/mL.
**p<0.01 vs DOX-hyd-PEG-FA NPs+FA; ##p<0.01 vs control, n = 5.
Figure 13.
In vivo antitumor activity of DOX-hyd-PEG-FA NPs.
Athymic nude mice xenografted with KB cells were intravenously injected with different doses of DOX-hyd-PEG-FA NPs (2.5, 5 and 10 mg/kg equivalent DOX) and one dose of DOX (5 mg/kg) every 7 day (days 1, 7, and 14). Panel A-tumor volume changes of tumor bearing mice, *P<0.05 vs free DOX, #P<0.05 vs 5 mg/kg DOX-hyd-PEG-FA NPs, n = 3. Panel B-body weight changes in tumor bearing nude mice, *P<0.05 vs free DOX, n = 3. Panel C-survival curve of tumor bearing mice. *P<0.05 vs free DOX, n = 3.
Table 2.
Cytotoxicity for A549 cells, KB cells and HepG2 cells treated with DOX, DOX-hyd-PEG-FA NPs, DOX-hyd-PEG NPs, DOX-ami-PEG-FA NPsz and DOX-ami-PEG NPs.
Table 3.
Statistic analysis of survival of tumor bearing mice.
Figure 14.
The DOX distribution in tumor bearing nude mice after intravenous injection of either free DOX (panel A) or DOX-hyd-PEG-FA NPs (panel B).
**P<0.01 vs free DOX group in heart tissue at the same time point; ##P<0.01 vs free DOX group in tumor tissue at the same time point; &P<0.05, &&P<0.01 vs free DOX group in blood at the same time point. n = 5.
Figure 15.
Histological sections of heart from tumor bearing nude mice.
Haematoxylin and eosin stain. Panel A-negative control mice (tumor bearing mice treated with normal saline). Panel B-doxorubicin treated mice (5 mg/kg), degenerative myofibrils (arrows) are seen. Panel C-DOX-hyd-PEG-FA NPs treated mice (equivalent dose of doxorubicin:5 mg/kg).