Vascular-targeted photothermal therapy of an orthotopic murine glioma model

Abstract

Aim: To develop nanoshells for vascular-targeted photothermal therapy of glioma.

Materials & methods: The ability of nanoshells conjugated to VEGF and/or poly(ethylene glycol) (PEG) to thermally ablate VEGF receptor-2-positive endothelial cells upon near-infrared laser irradiation was evaluated in vitro. Subsequent in vivo studies evaluated therapy in mice bearing intracerebral glioma tumors by exposing tumors to near-infrared light after systemically delivering saline, PEG-coated nanoshells, or VEGF-coated nanoshells. The treatment effect was monitored with intravital microscopy and histology.

Results: VEGF-coated but not PEG-coated nanoshells bound VEGF receptor-2-positive cells in vitro to enable targeted photothermal ablation. In vivo, VEGF targeting doubled the proportion of nanoshells bound to tumor vessels and vasculature was disrupted following laser exposure. Vessels were not disrupted in mice that received saline. The normal brain was unharmed in all treatment and control mice.

Conclusion: Nanoshell therapy can induce vascular disruption in glioma.

Figure 1. Nanoshell characterization

(A) Scanning electron microscopy confirmed nanoshells had a uniform size distribution. (B) Silver staining of proteins separated by gel electrophoresis confirmed VEGF conjugation to orthopyridyl disulfide–PEG–N-hydroxysuccinimide (2000 Da), with different bands of increased molecular weight appearing based on the number of PEG chains attached. (C) An ELISA revealed approximately 100 VEGF molecules bound to each NS; background on PEG-NS controls was minimal. (D) Upon functionalization, nanoparticle hydrodynamic diameter increased and zeta-potential magnitude decreased. NS: Nanoshell; PEG: Polyethylene glycol; PEG-NS: Polyethylene glycol-coated nanoshells; VEGF-NS: VEGF-coated nanoshells.

Figure 2. In vitro assessment of photothermal therapy mediated by VEGF-coated nanoshells

Mile sven 1 endothelial cells that overexpress VEGF receptor (VEGFR-2) were incubated with saline, PEG-NS or VEGF-NS prior to darkfield microscopy and 808-nm laser irradiation (6 W/cm², 3 min). (A) Darkfield microscopy proves that VEGF-NS bind mile sven 1 cells that express VEGFR-2, while PEG-NS do not. NSs appear red against the blue cell background. (B) Fluorescence microscopy reveals that only cells targeted with VEGF-NSs experience loss in viability upon laser irradiation. Live cells fluoresce green (calcein acetoxymethyl ester) and dead cells fluoresce red (ethidium homodimer-1). PEG-NS: Polyethylene glycol-coated nanoshells; VEGF-NS: VEGF-coated nanoshells.

Figure 3. Distribution of VEGF-coated and polyethylene glycol-coated nanoshells in mice with intracerebral tumors

(A) Gold content in various organs was determined by inductively coupled plasma–mass spectrometry. Data depicts mean ± standard deviation. No standard deviation is shown for the heart in the VEGF-NS group at 24 h because n = 2 in this group. (B) Nanoshell distribution to the tumor and normal brain is shown with an expanded y-axis. ^†Significant versus all normal brain groups. ^‡Significant versus both 6-h tumor groups; analysis of variance with post hoc Tukey. PEG-NS: Polyethylene glycol-coated nanoshells; VEGF-NS: VEGF-coated nanoshells.

Figure 4. Visualization of nanoshell distribution

Accumulation of PEG-NS and VEGF-NS in various organs was observed with darkfield microscopy (columns one and three). Fluorescence images (columns two and four) from the same fields of view show CD31 (green), nuclei (blue), and some tissue autofluorescence (orange). Generally, nanoshells are found in close proximity to blood vessels. PEG-NS: Polyethylene glycol-coated nanoshells; VEGF-NS: VEGF-coated nanoshells.

Figure 5. Assessment of nanoshell proximity to tumor vasculature

(A & B) To evaluate the targeting effect, images of nanoshells (red, derived from darkfield microscopy) were merged with fluorescence images from the same region in the tumor. Three representative areas, in which nuclei are blue (4′,6-diamidino-2-phenylindole), endothelial cells are green (CD31), and red blood cells are orange (autofluorescence) are shown for (A) VEGF-NSs and (B) PEG-NSs. Generally, VEGF-NSs remain closer to vessels than PEG-NSs. (C) Quantification of nanoshell distance from blood vessels confirmed that targeting with VEGF increased the adherence of nanoshells to tumor vessels. ^†Significant versus PEG-NS >10 μm; analysis of variance with post hoc Tukey. ^‡Significant versus PEG-NS <0 μm. ^§Significant versus VEGF-NS >0 μm. PEG-NS: Polyethylene glycol-coated nanoshells; VEGF-NS: VEGF-coated nanoshells.

Figure 6. Intravital microscopy reveals changes in tumor vasculature following treatment

(A) Changes in tumor signal and vessel morphology were qualitatively evaluated by comparing intravital microscopy images acquired on the day the laser was applied and 6 days later. The mouse treated with saline shows increasing tumor signal and signs of vessel maturation, while the mouse treated with VEGF-NS shows a stable tumor signal and signs of vessel disruption. (B) Quantification of vessel density from intravital microscopy images highlights the treatment effect. Mice that received saline exhibited a mean increase in vessel density of 18% over 3 days after laser treatment (green bar) while mice that received VEGF-NS experienced a 24% decrease in vessel density (turquoise bar). Data show mean ± standard error. These differences were significant at the 95% CI. *p = 0.025; student’s t-test. VEGF-NS: VEGF-coated nanoshells.

Figure 7. Histological evaluation of vascular-targeted photothermal therapy

Mice that received saline, PEG-NSs, or VEGF-NSs were sacrificed immediately after laser exposure to evaluate the effects of vascular-targeted photothermal therapy on the tumor and the normal brain with hematoxylin and eosin staining. No signs of thermal damage were observed in the normal brain for all groups nor in the tumor for mice exposed to saline. Comparatively, mice exposed to VEGF-NSs and PEG-NSs showed evidence of vessel dilation and hemorrhaging within the tumor.