Radiolabeled Angiogenesis-Targeting Croconaine Nanoparticles for Trimodality Imaging Guided Photothermal Therapy of Glioma

Abstract

To meet the criteria of effective theranostics, biocompatible nanomedicine endowing intrinsic therapeutic and imaging properties have gained extraordinary momentum. In this study, an ultra-stable near-infrared (NIR) dye croconaine (CR780) was engineered with arginine-glycine-aspartic acid (RGD) peptide and polyethylene glycol (PEG), which was then self-assembled into uniform nanoparticles (NPs). These RGD-CR780-PEG5K assemblies were radiolabeled with ¹²⁵I through a facile standard Iodo-Gen method. The resulting [¹²⁵I]RGD-CR780-PEG5K NPs showed effective accumulation in α_vβ₃ integrin expressing glioblastoma, as evidenced by single photon emission computed tomography (SPECT)/CT and NIR fluorescence imaging. More importantly, high-resolution photoacoustic imaging revealed that these NPs selectively targeted to angiogenic tumor vessels. With the favorable tumor selective accumulation and high photothermal conversion efficiency, the [¹²⁵I]RGD-CR780-PEG5K NPs allowed thorough tumor ablation and inhibition of tumor relapse at a relatively low laser energy (0.5 W/cm²). Overall, this work offers a proper methodology to fabricate tumor-targeted multi-modal nanotheranostic agents, providing great opportunity for precision imaging and cancer therapy.

Keywords: SPECT/CT; angiogenesis; croconaine nanoparticles; glioma; photothermal therapy.

Figure 1

Schematic illustration of radiolabled angiogenesis-targeting croconaine nanoparticles ([¹²⁵I]RGD-CR780-PEG5K NPs) for tumor α_vβ₃ integrin multimodality imaging guided photothermal therapy.

Figure 2

(A) Real-time thermal imaging and (B) temperature changes of RGD-CR780-PEG5K, Tyr-CR780-PEG5K in aqueous solutions (30 μM) and PBS with laser irradiation (808 nm, 0.5 W/cm² for 20 min). (C) Emission spectra of RGD-CR780-PEG5K and Tyr-CR780-PEG5K in aqueous solutions (30 μM) excited at 760 nm. (D–F) Vis-NIR spectra (D), Fluorescent images (E) and quantification of corresponding intensities of RGD-CR780-PEG5K and Tyr-CR780-PEG5K at 30 μM before and after laser irradiation (808 nm, 0.5 W/cm² for 20 min). (G–H) PA images (G) and corresponding amplitude (H) of RGD-CR780-PEG5K and Tyr-CR780-PEG5K at 30 μM. The data are shown as mean ± SD (n = 3).

Figure 3

(A) Subcellular localization of Tyr-CR780-PEG5K, RGD-CR780-PEG5K and RGD-CR780-PEG5K + free c(RGDyK) (block) (30 μM). Co-localization of the NIR dyes (Ex = 633 nm) with DAPI at 4 h in U87MG cells as imaged by confocal microscope. (B) Uptake of [¹²⁵I]Tyr-CR780-PEG5K, [¹²⁵I]RGD-CR780-PEG5K and [¹²⁵I]RGD-CR780-PEG5K + free c(RGDyK) (block) (37 KBq) by U87MG cells at different times. (C–D) Fluorescence images of calcein AM/PI co-stained U87MG cells (C) and Cell viability determined by MTT assay (D) after incubation with Tyr-CR780-PEG5K, RGD-CR780-PEG5K and RGD-CR780-PEG5K + free c(RGDyK) (block) (30 μM) for 4 h with or wihtout being exposed to 808 nm laser at 0.5 W/cm² for 5 min. The data are shown as mean ± SD (n = 3), ** P < 0.01, *** P < 0.001, compared with non-irradiated group.

Figure 4

(A–B) Whole-body NIR fluorescent imaging (A) and correspondingly tumor-to-muscle ratios (B) of U87MGtumor-bearing mice after intravenous (i.v.) injection of Tyr-CR780-PEG5K, RGD-CR780-PEG5K and RGD-CR780-PEG5K + free c(RGDyK) (block). Images were acquired at indicated time points and the color bar indicates radiant efficiency (low, 2.5×10⁶; high, 4.80×10⁶). Red circles were used to indicate tumors. (C–D) PA imaging (C) and PA intensities (D) of tumor tissues in U87MG tumor-bearing mice at 6 h after injection. n = 4 per group, ** P < 0.01, *** P < 0.001.

Figure 5

In vivo behavior of the probes. (A) SPECT/CT imaging of tumor-bearing mice 6 h after intravenous injection with [¹²⁵I]Tyr-CR780-PEG5K, [¹²⁵I]RGD-CR780-PEG5K, or [¹²⁵I]RGD-CR780-PEG5K probes plus free RGD peptide (100 μL, 0.15 mM), at a radiation dose of 18.5 MBq. (B) Biodistributions of the probes 6 h after injection. (C) Blood clearance profile of [¹²⁵I]RGD-CR780-PEG5K after intravenous injection. The data are shown as mean ± SD (n = 4), * P < 0.05, ** P < 0.01.

Figure 6

In vivo PTT effect. (A) Thermal images of U87MG tumor-bearing mice i.v. treated with 100 μL PBS, Tyr-CR780-PEG5K, RGD-CR780-PEG5K or RGD-CR780-PEG5K + c(RGDyK) (block, 200 μg) (2 mM) and illuminated with 808 nm laser (0.5 W/cm², 10 min) at 6 h post-injection. (B) Quantitative analysis of temperature changes in tumor area. (C) U87MG tumor growth rate in each groups after indicated treatments. Tumor volumes were normalized to their initial size (n = 5 per group). (D) Survival curves of tumor-bearing mice after various treatments. RGD-CR780-PEG5K + laser group showed 100% survival over 40 days. (E) Representative images of mice bearing U87MG tumors after treatments. The data are shown as mean ± SD (n = 3), ** P < 0.01.

Scheme 1

Synthesis of [¹²⁵I]Tyr-CR780-PEG5K (A) and [¹²⁵I]RGD-CR780-PEG5K (B). Reagents: (a), (c) EDC, NHS, DIPEA, CH₂Cl₂, 0 °C-r.t., 4 h, 85%; (b), (d) 1) [¹²⁵I]-NaI, Iodo-Gen, r.t., 3.5 min, 2) Na₂S₂O₅