Figure 1.
SP5-2 enhances internalization of liposomes.
(A) Fluorescence microscopy was used to visualize H460 cells after incubation with either SP5-2-liposomal SRB (SP5-2-LS), MP5-2-LS or LS at the indicated temperatures. Nuclear staining was performed using DAPI (Scale bar: 30 µm). Internalization studies of SP5-2-LD and LD in H460 cells were incubated with 10 µg/ml (B) or 2.5 µg/ml (C) drugs at 37°C, respectively. Doxorubicin uptake by the cells was quantified at the indicated times, following the removal of surface-bound liposomal drugs.
Figure 2.
Transmission electron microscopy reveals SP5-2-mediated internalization of liposomal doxorubicin in H460 cells.
(A) Transmission electron micrographs of H460 cells at 37°C, following a 10-minute incubation with SP5-2-LD (final doxorubicin concentration of 2 µg/ml). Arrows indicate endosome-containing liposomes. (B) Percentage of cells containing internalized liposomes (positive rate), after incubation with either SP5-2-LD or MP5-2-LD. (C) The number of liposomes per vesicle were calculated using randomly selected endosomes in H460 cells (n = 30 in each group, P = 1.23×10−7).
Figure 3.
Verification of SP5-2 binding to a mouse (LL/2) and human (H460) lung cancer cell lines.
(A) Binding of peptide-conjugated Qdots to tumor cells in vitro is peptide-sequence specific. The ability of quantum dots labeled with SP5-2 and MP5-2 to bind to the surface of LL/2 cells was examined (Scale bar, 50 µm). Nuclear staining was performed using DAPI. (B) LL/2 cells were incubated with SP5-2 FITC-conjugated (FITC-SP5-2) or MP5-2 FITC-conjugated (FITC-MP5-2) peptides at increasing concentrations. The FITC-SP5-2 treated LL/2 cells showed increasing flurescence intensity at increasing peptide concentrations, but no such correlation was observed in FITC-MP5-2 treated cells. (C) H460 cells were seeded in 96-well ELISA plates. Biotinylated SP5-2 (B-SP5-2) and biotinylated MP5-2 (B-MP5-2) were used to test the peptide binding assay. (D) Peptide competitive inhibition assay with the unlabeled SP5-2 peptide. The black bars showed the binding inhibition of the biotinylated peptide (B-SP5-2, 10 µg/ml) by the unlabeled SP5-2 peptide (from 22 to 600 µg/ml). The gray bars showed the binding inhibition of B-SP5-2 by the MP5-2 peptide. The statistic value was compared to control peptide. **, p value <0.01; ***, p value <0.005.
Figure 4.
SP5-2-LD increases therapeutic efficacy in a syngenic animal model.
(A) C57BL/6 mice bearing syngenic LL/2 xenografts were injected i.v. with PC5-2 (phage displaying SP5-2) or control phages, and phage titers were examined in tumor tissue and the indicated organs. (B) Eight-week-old C57BL/6 mice bearing LL/2 xenografts were injected i.v. with different doxorubicin formulations twice a week for 17 days. Mice were treated with either 1 mg/kg or 2 mg/kg of SP5-2-LD, MP5-2-LD, LD, FD or PBS. In the 1 mg/kg treatment of doxorubicin, tumor size (B) and body weight (C) were measured at the time point of injection and at the indicated times thereafter. (D, E) indicate the tumor size and body weight of 2 mg/kg treatment, respectively. All data shown are on the mean averages of six mice per group. (*, p value <0.05.)
Figure 5.
SP5-2-LD enhances survival in a metastatic tumor animal model.
C57BL/6 mice were injected i.v. with 1×105 LL/2 cells, incubated for 24 hours, and then treated with different doxorubicin formulations via i.v. injections twice a week. Mice were treated with either 1 mg/kg or 2 mg/kg of SP5-2-LD, MP5-2-LD, LD, FD or PBS (n = 8). The survival rate is shown in (A, C) for 1 and 2 mg/kg treatment, respectively. Body weight is shown in (B, D) for 1 and 2 mg/kg treatment, respectively. (**, p value <0.01; ***, p value <0.005.)
Figure 6.
SP5-2-LD enhances survival and reduces side-effects in an orthotopic tumor model.
The lungs of SCID mice were injected with 5×105 H460 cells. Mice were treated with SP5-2-LD, MP5-2-LD, LD, FD or PBS (n = 6, 1 mg/kg, twice a week) five days after injection. Median overall survival is shown in (A). Body weight is shown in (B). (*, p value <0.05; **, p value <0.01).
Figure 7.
Biodistribution of doxorubicin in mice treated with different anti-cancer drug formulations.
Doxorubicin formulations were injected into the tail vein of H460 tumor-bearing mice. Various organs were collected at the indicated time points, and the doxorubicin concentration was determined (bars, S.D.). The concentrations in each organ are normalized to that in tumor at the equivalent time point, and are presented as the average of three experiments. The biodistribution of free doxorubicin (A), liposomal doxorubicin (LD) (B), MP5-2-LD (C), and SP5-2-LD (D) in each organ of the treated mice are shown. (E) Comparison of doxorubicin accumulation in tumor tissue. Tumor tissues were collected, homogenized, and quantified for fluorescence. (F) Cellular nuclei were isolated from the tumor tissues, and the concentration of nuclear doxorubicin was determined by measuring the intrinsic auto-fluorescence signal of doxorubicin. All data are the mean averages of data from three mice per group.
Table 1.
Tumor pharmacokinetics of free doxorubicin versus liposomal doxorubicin formulations.