Endometriosis-targeted MRI imaging using bevacizumab-modified nanoparticles aimed at vascular endothelial growth factor

Abstract

Endometriosis is a tumor-like disease with high recurrence. In this case, the accurate imaging-based diagnosis of endometriosis can help clinicians eradicate it by improving their surgical plan. However, although contrast agents can improve the visibility of the tissue of interest in vivo via magnetic resonance imaging (MRI), the lack of biomarkers in endometriosis hinders the development of agents for its targeted imaging and diagnosis. Herein, aiming at the enriched vascular endothelial growth factor (VEGF) in endometriosis, we developed a targeting MRI contrast agent modified with bevacizumab, i.e., NaGdF₄@PEG@bevacizumab-Cy5.5 nanoparticles (NPBCNs), to detect endometriosis. NPBCNs showed negligible cytotoxicity and high affinity towards VEGF in endometrial cells in vitro. Furthermore, NPBCNs generated a strong signal enhancement in vivo in endometriosis lesions in rats in T₁-weighted images via MRI at 3 days post-injection, as confirmed by the histopathological staining results and fluorescence imaging on the same day. Our approach can enable NPBCNs to target endometriosis effectively, thus avoiding missed diagnoses.

Fig. 1. Schematic illustration of the facile synthesis of NaGdF₄@PEG@bevacizumab–Cy5.5 nanoparticles (NPBCNs) and their biomedical application as a contrast agent for MRI and fluorescence imaging for the diagnosis of endometriosis.

Fig. 2. Characterization of NaGdF₄@PEG@bevacizumab–Cy5.5 nanoparticles (NPBCNs). (a) Transmission electron microscopy (TEM) images of NaGdF₄ nanoparticles and NPBCNs. (b) X-ray diffraction (XRD) pattern of NHS–PEG–cholesterol, NPBCNs, NaGdF₄@PEG–NHS–Cy5.5 nanoparticles (NPCNs) and NaGdF₄ nanoparticles. (c) Fourier transformation infrared (FTIR) spectra of NaGdF₄ nanoparticles, NPCNs, NPBCNs. (d) T₁-Weighted MR imaging of NPBCNs at different concentrations of Gd and the T₁ relaxivity NPBCNs.

Fig. 3. Representative confocal laser scanning microscopy (CLSM) images and analysis of cellular uptake of nanoparticles in hEM15A endometrial cell line in vitro. (a) hEM15A cells incubated with NaGdF₄@PEG@bevacizumab–Cy5.5 nanoparticles (NPBCNs) (upper) and NaGdF₄@PEG–NHS–Cy5.5 nanoparticles (NPCNs) (middle). After blocking the VEGF signaling pathway with free bevacizumab, hEM15A cells were incubated with NPBCNs (bottom). (b) Quantitative analysis of mean fluorescence intensity in hEM15A cells. ****, p < 0.001; ns, non-significant.

Fig. 4. Targeted imaging performance of NaGdF₄@PEG@bevacizumab–Cy5.5 nanoparticles (NPBCNs) in endometriosis rats. (a) MR-signal intensity of the endometriosis lesion in the targeted group and non-targeted group with prolonged duration after intravenous administration (*, p < 0.05). (b) Representative T₁-weighted images of rats with endometriosis in the targeted group before and after intravenous injection of NPBCNs. (c) Representative T₁-weighted images of rats with endometriosis in the non-targeted group before and after intravenous injection NaGdF₄@PEG–NHS–Cy5.5 nanoparticles (NPCNs). (d) Fluorescence image of the targeted group and the non-targeted group and NPCNs on 3 d post-injection (H = high and L = low).

Fig. 5. Histological assessment of endometriosis lesion in the targeted group. (a) Representative histologic image (400×) from of models with endometriosis. (b) Representative colocalization images of endometriosis lesions from rats injected with NaGdF₄@PEG@bevacizumab–Cy5.5 nanoparticles (NPBCNs). Cy5.5 channel: NPBCNs; Hoechst channel: cell nuclei and FITC channel: the immunofluorescence staining for the makers for ectopic endometrial cells [cyclooxygenase-2 (COX2) oxytocin receptor (OTR), nerve growth factor (NGF) and PGP9.5].