Figure 1.
Combination treatment of ALN and TNP-470 has synergistic inhibitory effect on proliferation of endothelial cells in vitro.
(A) ALN (closed squares) and TNP-470 (open squares) had synergistic effect on HUVEC proliferation when combined as ALN+TNP-470 0.01 pM (closed triangles) and TNP-470+ALN 10 µM (open triangles). (B) Classic isobologram of ALN−TNP-470 combination treatment. IC70 (closed squares), IC50 (closed triangles) and IC30 (closed circles) of ALN and TNP-470. Dashed circles, represent synergism areas of combination. Tables represent the combination index values of each IC of combination treatments I and II. Drug concentration in nM is presented on a logarithmic scale. Data represent mean±s.d.
Figure 2.
Characterization of HPMA copolymer-Gly-Gly-Pro-Nle-ALN-TNP-470 conjugates.
SEC profiles of polymerization I (classical) compound (A) and polymerization II (RAFT) compound (B). Hydrodynamic diameter size distribution (C) of polymerization I compound (gray bars) and polymerization II compound (white bars). (D) Binding kinetics of HPMA copolymer-ALN-TNP-470 conjugate to HA.
Figure 3.
Subcellular trafficking of FITC-labeled HPMA copolymer-ALN-TNP-470 conjugate in HUVEC and Saos-2 human osteosarcoma cells by confocal microscopy.
(A) Single XY plane imaging of the conjugate (green) with PI (red) nuclei staining showed cytoplasmatic accumulation of the conjugate. Cellular uptake analysis of the conjugate by HUVEC cells (B) 5.7 µm Z-stack of 28 slices and (C) XZ image slice revealed similar conjugate and nuclei focal plane localization. Multi-channel overlay of (G) HUVEC and (K) Saos-2 cells stained with phalloidin (red) for actin filaments (D, H) and DAPI (blue) for nuclei (F, J) 12 h post incubation with the conjugate (green) (E, I) showed cellular localization of the conjugate mostly around the nuclei. The FITC-labeled conjugate (green) (L, P) colocalized with clathrin-coated vesicles labeled with transferrin (red) (M, Q) in HUVEC (M, N) and Saos-2 (Q, R) cells. Histograms of overlay images revealed a ratio of conjugate/transferrin of 82% in HUVEC (O) and 71% in Saos-2 (S) cells pointing at the cellular uptake of the conjugate via the lysosomotropic pathway. Scale bars represent (A–K) 5 µm and (L–S) 20 µm.
Figure 4.
Inhibition of proliferation of endothelial, Saos-2 and MG-63-Ras osteosarcoma cells by HPMA copolymer-ALN-TNP-470 conjugate.
Free (open squares/triangles/diamonds) and polymer-conjugated ALN and TNP-470 (closed squares/triangles/diamonds) had similar effect on cell proliferation exhibiting retention of activity following polymer conjugation. Free and conjugated ALN and TNP-470 inhibited ECGS-induced HUVEC proliferation at IC50 of 0.7 and 1 nM respectively. Free and conjugated drugs had cytotoxic effect on these cells at doses >1 and 10 nM respectively. The inhibition of endothelial proliferation by the conjugate was reduced significantly (IC50 = 4200 nM) in the presence of cathepsin K inhibitor III (open circles). Proliferation of Saos-2 and MG-63-Ras cells was inhibited by free and conjugated ALN and TNP-470 at higher concentrations with IC50 of 30 µM and 10 µM respectively. The solid line represents the proliferation of growth factor-induced cells and the dotted line represents the cell proliferation in the absence of ECGS. Data represent mean±s.d. * P<0.05, ** P<0.03, *** P<0.05 compared with control.
Figure 5.
Inhibition of VEGF-induced migration and capillary-like tube formation by HPMA copolymer-ALN-TNP-470 conjugate.
Free (dotted bars) and polymer-conjugated ALN and TNP-470 (gray bars) inhibited both (A) VEGF-induced HUVEC migration; and (B, C) capillary-like tube formation of HUVEC, in a dose-dependent manner. Scale bars represent 100 µm. Drug concentration in nM is presented on a logarithmic scale. All P values are two-sided (analysis of variance). Data represent mean±s.d. * P<0.05, ** P<0.03, *** P<0.05 compared with control.
Figure 6.
HPMA copolymer-ALN-TNP-470 conjugate reduces vascular hyperpermeability in mouse skin capillaries and inhibits MG-63-Ras human osteosarcoma growth.
(A) Diminished dye is observed in mice treated with free or conjugated ALN and TNP-470 compared to untreated control group. Scale bar represents 10 mm. (B) Extracted dye content quantification by measurement at 620 nm. (C) Intravital non-invasive fluorescence imaging of mCherry-labeled MG-63-Ras tumor-bearing mice treated with free or conjugated ALN and TNP-470. (D) Antitumor effect of free (open triangles) or conjugated (closed triangles) ALN and TNP-470 on MG-63-Ras human osteosarcoma tumor growth compared to vehicle-treated group (closed squares) and dissected tumors images. Scale bar represents 10 mm. All P values are two-sided. On day 19 HPMA copolymer-ALN-TNP-470 conjugate inhibited tumor growth by 96% (P = 0.001) compared with 45% (P = 0.012) of free ALN and TNP-470. Data represent mean±s.d. * P<0.05, ** P<0.03, *** P<0.05 compared with control. (E) Whole-mount confocal microscopy of mCherry-labeled MG-63-Ras human osteosarcoma tumors dissected from mice treated with FITC-labeled HPMA copolymer-ALN-TNP-470 conjugate. Scale bar represents 25 µm (n = 5 mice per group). (F) H & E and CD34 staining of control, combination- and conjugate-treated MG-63-Ras osteosarcomas inoculated s.c. in mice. (G) Dissected organs of mice treated with FITC-labeled HPMA copolymer-ALN-TNP-470 conjugate were imaged by the Maestro system showing greater intensity of FITC-fluorescence spectrum (composed images of unmixed multispectral cubes) in bone tissues then in the spleen, heart, lungs, kidneys and liver. Green represent FITC-labeled HPMA copolymer-ALN-TNP-470 conjugate spectrum.