Targeted chemotherapy for subcutaneous and orthotopic non-small cell lung tumors with cyclic RGD-functionalized and disulfide-crosslinked polymersomal doxorubicin

Abstract

Lung cancer, with its high mortality and increasing morbidity, has become one of the most lethal malignancies worldwide. Here, we developed cyclic RGD peptide-directed and disulfide-crosslinked polymersomal doxorubicin (cRGD-PS-Dox) as a targeted chemotherapy for human non-small cell lung cancer (NSCLC). Notably, cRGD-PS-Dox exhibited a high Dox loading (15.2 wt.%), small hydrodynamic diameter (96 nm), superb stability, prominent targetability to α_vβ₃ integrin overexpressing A549 human lung cancer cells, and rapid release of the drug into nuclei, leading to a significantly improved antitumor activity compared with the control groups, i.e., PS-Dox and Lipo-Dox (a liposome injection employed in clinical settings). The pharmacokinetic and biodistribution results for cRGD-PS-Dox revealed similar elimination half-lives but two-fold enhanced tumor accumulation compared with PS-Dox and Lipo-Dox. Intriguingly, cRGD-PS-Dox effectively suppressed the growth of A549 lung tumors in both subcutaneous and orthotopic models with minimal adverse effects at a Dox dose of 12 mg/kg, leading to significant survival benefits compared with PS-Dox and Lipo-Dox. This α_vβ₃ integrin-targeting multifunctional polymersomal doxorubicin is highly promising for targeted chemotherapy of human NSCLC.

Fig. 1

a Illustration of cRGD-directed polymersomal doxorubicin (cRGD-PS-Dox) for active targeting chemotherapy of lung tumor xenografts in mice; b DLS measurement and TEM photograph of cRGD-PS; c UV absorbance of cRGD-PS before and after crosslinking; d In vitro Dox release in PB with or without GSH (10 mM) at 37 °C

Fig. 2

a Cytotoxicity of blank cRGD-PS to A549 cells following 48 h incubation. b Viability of A549 cells after a 4 h incubation with cRGD-PS-Dox plus 44 h culture in fresh media. c Flow cytometry of A549 cells after a 4 h incubation with different formulations (10.0 μg/mL Dox·HCl). d CLSM of A549 cells treated with different formulations for 4 h (10.0 μg/mL Dox·HCl) and further cultured in fresh media for 4 h. The cell nuclei and cytoskeleton were stained by DAPI and phalloidin-FITC, respectively. Scale bars: 25 μm

Fig. 3

a In vivo pharmacokinetics of cRGD-PS-Dox, PS-Dox and Lipo-Dox in tumor-free mice. b In vivo fluorescence images of mice bearing subcutaneous A549 tumors after iv injection of Cy7-labeled cRGD-PS or PS. c Ex vivo fluorescence images of tumors and major organs isolated at 12 h post-injection. d Quantitative drug biodistribution in tumors and major organs. *p < 0.05 based on one-way ANOVA and Tukey multiple comparisons tests. For a and d, the Dox·HCl concentration was measured by fluorescence spectroscopy and are presented as the mean ± SD (n = 3)

Fig. 4

Antitumor activity of cRGD-PS-Dox and PS-Dox in nude mice bearing subcutaneous A549 tumors. The drug was given on day 0, 4, 8, and 12 (Dox dose: 6 or 12 mg/kg, in 200 µL PBS). Lipo-Dox and PBS were used as controls. a Tumor volumes over 20 days. b Body weight changes over 20 d. c Photographs of tumor blocks collected on day 20. d Tumor inhibition rate on day 20. *p < 0.05, **p < 0.01 and ***p < 0.001 based on one-way ANOVA and Tukey multiple comparisons tests

Fig. 5

In vivo antitumor activity of cRGD-PS-Dox, PS-Dox, and Lipo-Dox in nude mice bearing orthotopic A549 tumor xenografts at a Dox dose of 6 or 12 mg/kg in 200 µL PBS administered on day 0, 4, 8, and 12. a Bioluminescence images of mice over time. b Bioluminescence intensity of mice. One-way ANOVA and Tukey multiple comparisons tests, *p < 0.05 and ***p < 0.001. c Body weight of mice over 16 days. d Survival curves of mice

Fig. 6

Ex vivo luminescence images of major organs from mice bearing orthotopic A549-Luc tumors on day 16. I. cRGD-PS-Dox@12 mg/kg, II. cRGD-PS-Dox@6 mg/kg, III. PS-Dox@6 mg/kg, IV. Lipo-Dox@6 mg/kg, V. PBS