Enhancing Drug Delivery for Overcoming Angiogenesis and Improving the Phototherapy Efficacy of Glioblastoma by ICG-Loaded Glycolipid-Like Micelles

Abstract

Background: Phototherapy is a potential new candidate for glioblastoma (GBM) treatment. However inadequate phototherapy due to stability of the photosensitizer and low target specificity induces the proliferation of neovascular endothelial cells for angiogenesis and causes poor prognosis.

Methods: In this study, we constructed c(RGDfk)-modified glycolipid-like micelles (cRGD-CSOSA) encapsulating indocyanine green (ICG) for dual-targeting neovascular endothelial cells and tumor cells, and cRGD-CSOSA/ICG mediated dual effect of PDT/PTT with NIR irradiation.

Results: In vitro, cRGD-CSOSA/ICG inhibited cell proliferation and blocked angiogenesis with NIR irradiation. In vivo, cRGD-CSOSA/ICG exhibited increased accumulation in neovascular endothelial cells and tumor cells. Compared with that of CSOSA, the accumulation of cRGD-CSOSA in tumor tissue was further improved after dual-targeted phototherapy pretreatment. With NIR irradiation, the tumor-inhibition rate of cRGD-CSOSA/ICG was 80.00%, significantly higher than that of ICG (9.08%) and CSOSA/ICG (42.42%). Histological evaluation showed that the tumor vessels were reduced and that the apoptosis of tumor cells increased in the cRGD-CSOSA/ICG group with NIR irradiation.

Conclusion: The cRGD-CSOSA/ICG nanoparticle-mediated dual-targeting phototherapy could enhance drug delivery to neovascular endothelial cells and tumor cells for anti-angiogenesis and improve the phototherapy effect of glioblastoma, providing a new strategy for glioblastoma treatment.

Keywords: angiogenesis; dual-targeting; glioblastoma; glycolipid-like micelles; phototherapy.

Figure 1

Schematic diagram of phototherapy effect with dual-targeting drug delivery system. The cRGD-CSOSA/ICG nanoparticles are absorbed by tumor cells and neovascular endothelial cells rapidly, and PDT/PTT double efficacy occurs with NIR irradiation to induce cell apoptosis and necrosis for GBM therapy.

Figure 2

Characterization of cRGD-CSOSA. (A) ¹H-NMR spectra of CSO, SA, CSOSA, cRGD, and cRGD-CSOSA in D₂O. (B) CMC values of CSOSA and cRGD-CSOSA. (C) TEM images of CSOSA/ICG. (D) TEM images of cRGD-CSOSA/ICG. (E) The stability of ICG-loaded nanoparticles. ***p < 0.001.

Figure 3

Photothermal conversion efficiency and ROS detection in vitro (A) Photothermal conversion efficiency of ICG, CSOSA/ICG and cRGD-CSOSA/ICG with the NIR irradiation (2 W/cm²). (B) The production of extracellular ROS by NIR irradiation (2 W/cm²). (C) The production of intracellular ROS mediated by NIR irradiation (2 W/cm², 3 min). (D) The median fluorescence intensity of intracellular ROS produced by U87 MG cells. **p < 0.01, ***p < 0.001.

Figure 4

Dual-targeting uptake of cRGD-CSOSA. U87 MG cells (A) and HUVECs (B) were incubated with CSOSA and cRGD-CSOSA for 1 and 4 h, respectively. (C) The median fluorescence intensity of U87 MG cells were incubated with different micelles for 1 and 4 h, respectively. (D) The median fluorescence intensity of HUVECs were incubated with different micelles for 1 and 4 h, respectively. (Data are represented as mean ± SD, n=3). **p < 0.01, ***p < 0.001.

Figure 5

The internalization mechanism of cRGD-CSOSA, after incubation with cRGD at 0 μM, 1 μM, 10 μM, 20 μM for 2 h, U87 MG cells (A) and HUVECs (B) were incubated with cRGD-CSOSA for 2 h.

Figure 6

In vitro cytotoxicity and anti-angiogenesis. (A) In vitro cytotoxicity of ICG, CSOSA/ICG and cRGD-CSOSA/ICG with the NIR irradiation (2 W/cm², 3 min). (B) The effects of ICG-loaded nanoparticles on proliferation of HUVECs with the NIR irradiation (2 W/cm², 3 min). (C) The ability of ICG-loaded nanoparticles to anti-angiogenesis with the NIR irradiation (2 W/cm², 3 min). **p < 0.01, ***p < 0.001.

Figure 7

In vivo Imaging and bio-distribution. (A) In vivo Imaging and distribution of ICG, CSOSA/ICG and cRGD-CSOSA/ICG after i.v. injection for 12, 24, 48, 72 and 96 h. (B) The fluorescent imaging of tumor tissues and main organs. (C) The distribution of CSOSA and cRGD-CSOSA in tumor tissue after i.v. injection for 24 h. (D) After phototherapy, the distribution of CSOSA and cRGD-CSOSA in tumor tissue.

Figure 8

In vivo antitumor activity. (A) In vivo infrared thermal imaging of ICG, CSOSA/ICG and cRGD-CSOSA/ICG after i.v. injection for 24 h (1 W/cm²). (B) Tumor volume changes (n=5). (C) Variations of body weight (n=5). (D) Final tumor weights (mean ± SD, n=5), **p < 0.01, ***p < 0.001.

Figure 9

Histological evaluation of U87 MG tumor xenografted nude mice model in four groups with NIR irradiation. (A) H&E images of major organs. (B) H&E images of tumor tissue. The arrows indicate the apoptotic cells. (C) Tumor blood vessel staining with CD 31 antibody (brown). (D) The induction of apoptosis in vivo studies on tumor tissues stained with C-Caspase3 antibodies (brown). (E) Cell proliferation evaluation by Ki67 staining (brown).