Optical and nuclear imaging of glioblastoma with phosphatidylserine-targeted nanovesicles

Abstract

Multimodal tumor imaging with targeted nanoparticles potentially offers both enhanced specificity and sensitivity, leading to more precise cancer diagnosis and monitoring. We describe the synthesis and characterization of phenol-substituted, lipophilic orange and far-red fluorescent dyes and a simple radioiodination procedure to generate a dual (optical and nuclear) imaging probe. MALDI-ToF analyses revealed high iodination efficiency of the lipophilic reporters, achieved by electrophilic aromatic substitution using the chloramide 1,3,4,6-tetrachloro-3α,6α-diphenyl glycoluril (Iodogen) as the oxidizing agent in an organic/aqueous co-solvent mixture. Upon conjugation of iodine-127 or iodine-124-labeled reporters to tumor-targeting SapC-DOPS nanovesicles, optical (fluorescent) and PET imaging was performed in mice bearing intracranial glioblastomas. In addition, tumor vs non-tumor (normal brain) uptake was compared using iodine-125. These data provide proof-of-principle for the potential value of SapC-DOPS for multimodal imaging of glioblastoma, the most aggressive primary brain tumor.

Keywords: PET; SapC-DOPS; glioblastoma; liposome; optical imaging.

Figure 1. Schematic representation of phenol-substituted, iodinated lipophilic dyes conjugated to tumor-targeted SapC-DOPS nanovesicles for dual imaging of glioblastoma

Tumor selectivity is determined by the affinity of SapC towards PS, a phospholipid sequestered in the inner leaflet of the plasma membrane in non-tumor cells, but markedly externalized upon neoplastic transformation.

Figure 2. Synthesis and characterization of phenol-substituted dye analogs

A. Synthetic scheme to prepare phenol substituted fluorescent analogs. B. MALDI-ToF spectrum of compound 2a. C. MALDI-ToF spectrum of compound 2b.

Figure 3. MALDI-ToF spectrum of compound 2a reacted with ¹²⁷I

Cold iodination with NaI (¹²⁷I) was carried out by dissolving compound 2a in TBA/PBS and reacting the mixture with NaI in Iodogen pre-coated tubes. Higher labeling efficiency was achieved using a 1:1 co-solvent ratio (50% TBA).

Figure 4. MALDI-ToF spectra of compound 2b reacted with ¹²⁷I

Iodination of compound 2b with NaI (¹²⁷I) was carried out by dissolving compound 2b in TBA/PBS and reacting the mixture with NaI in Iodogen pre-coated tubes. Higher labeling efficiency was achieved using a 1:3 co-solvent ratio (25% TBA).

Figure 5. Structure of SapC-DOPS nanovesicles

Under slightly acidic conditions, the hydrophobic protein SapC and the phospholipid dioleoyl-phosphatidylserine (DOPS) assemble into stable ~200 nm proteoliposomes, as seen by freeze-fracture electron microscopy. As depicted in the schematic illustration on the right, multimodal cancer imaging and therapy are possible by functionalization with contrast agents and radioligands.

Figure 6. Selective targeting of intracranial glioblastoma by SapC-DOPS conjugated with an iodinated fluorochrome

A. A mouse bearing a human glioblastoma xenograft (U87ΔEGFR-Luc cells) was injected (tail vein) with SapC-DOPS conjugated with cold-labeled, (¹²⁷I) phenolic 2a. 24 h later tumor bioluminescence (BLI) and compound 2a's fluorescence (right) were assessed in the excised brain, confirming colocalization. B. Mice bearing intracranial glioblastoma (TUMOR) or saline (SHAM) were injected (tail vein) with SapC-DOPS conjugated with ¹²⁵I-labeled phenolic 2a (5 ± 0.2 μCi). At different time points, tissues and organs were dissected and the incorporated radioactivity was measured and expressed as % injected dose (ID)/gram. Top graph shows brain activity; bottom graph shows thyroid organ activity. Tumor bearing mice: n = 7 (1 h); n = 6 (3 h); n = 2 (6 h); n = 6 (24 h). Sham: n = 4 (1, 3, 24 h); n = 2 (6 h). *, p < 0.05; **, p < 0.01 (t-test). C. microPET imaging of a glioblastoma in a mouse brain 24 h after administration of two i.v. injections (spaced 2 h apart) of 300 μl (50 μCi) SapC-DOPS-¹²⁴I· (2a) nanovesicles. A CT scan was acquired for anatomical co-registration and attenuation correction of the PET data. Imaging data was processed using Siemens IRW software (v4.1). Concurrent bioluminescence imaging (BLI) confirmed the presence of glioblastoma.