Targeting T1 and T2 dual modality enhanced magnetic resonance imaging of tumor vascular endothelial cells based on peptides-conjugated manganese ferrite nanomicelles

Abstract

Tumor angiogenesis plays very important roles for tumorigenesis, tumor development, metastasis, and prognosis. Targeting T1/T2 dual modality magnetic resonance (MR) imaging of the tumor vascular endothelial cells (TVECs) with MR molecular probes can greatly improve diagnostic sensitivity and specificity, as well as helping to make an early diagnosis of tumor at the preclinical stage. In this study, a new T1 and T2 dual modality nanoprobe was successfully fabricated. The prepared nanoprobe comprise peptides CL 1555, poly(ε-caprolactone)-block-poly(ethylene glycol) amphiphilic copolymer shell, and dozens of manganese ferrite (MnFe2O4) nanoparticle core. The results showed that the hydrophobic MnFe2O4 nanoparticles were of uniform spheroidal appearance and narrow size distribution. Due to the self-assembled nanomicelles structure, the prepared probes were of high relaxivity of 281.7 mM(-1) s(-1), which was much higher than that of MnFe2O4 nanoparticles (67.5 mM 1 s(-1)). After being grafted with the targeted CD105 peptide CL 1555, the nanomicelles can combine TVECs specifically and make the labeled TVECs dark in T2-weighted MR imaging. With the passage on, the Mn(2+) ions were released from MnFe2O4 and the size decreased gradually, making the signal intensity of the second and third passage of labeled TVECs increased in T1-weighted MR imaging. Our results demonstrate that CL-poly(ethylene glycol)-MnFe2O4 can conjugate TVECs and induce dark and bright contrast in MR imaging, and act as a novel molecular probe for T1- and T2-enhanced MR imaging of tumor angiogenesis.

Keywords: CD105; CL 1555; CL-PEG-MnFe2O4; TVECs; tumor angio genesis.

Figure 1

TEM characterization of MnFe₂O₄ NPs and PEG-b-PCL-MnFe₂CO₄ NMs. Notes: MnFe₂O₄ NPs were of spheroidal appearance and narrow size distribution (A) and the average size was 7.6±1.0 nm (B). The diameter of PEG-b-PCL-MnFe₂O₄ NMs was 146.7±25.9 nm (C and D). Inset of (A) is SAED of MnFe₂O4 NPs. Inset of (C) is an enlarged TEM of a single PEG-b-PCL-MnFe₂O₄ NM; magnification ×200,000. Abbreviations: NMs, nanomicelles; NPs, nanoparticles; PEG-b-PCL, polyethylene glycol-block-poly(ε-caprolactone); SAED, selected area electron diffraction; TEM, transmission electron microscopy.

Figure 2

HRTEM and EDS characterization of MnFe₂O₄ NPs. Notes: HRTEM image of a single MnFe₂O₄ NP showed that the spacing between the lattice fringes of planes (220) and (311) was around 0.257 nm (A) and 0.301 nm (B). The chemical composition of the nanocrystals was confirmed using EDS measurement. EDS (C) of the prepared NPs indicated the presence of Mn, Fe, and O, and the atomic ratio of Fe to Mn was around 2:1. Scale bar =10 nm. Abbreviations: EDS, energy dispersive spectrometer; HRTEM, high resolution transmission electron microscopy; NPs, nanoparticles.

Figure 3

Hydrodynamic diameter of MnFe₂O₄ NPs and NMs. Notes: The hydrodynamic diameter of MnFe₂O₄ dispersed in hexane was 10.3±1.2 nm (A) and that of PEG-b-PCL-MnFe₂O₄ and CL-PEG-b-PCL-MnFe₂O₄ was 162.6±28.9 nm (B) and 183.4±26.5 (C), respectively. Abbreviations: NMs, nanomicelles; NPs, nanoparticles; PEG-b-PCL, polyethylene glycol-block-poly(ε-caprolactone).

Figure 4

Hysteresis loop and T₂ relaxivity. Notes: The hysteresis loop of MnFe₂O₄ NPs (A) showed that the magnetization rose nonlinearly with the increase of applied magnetic field and no remaining net magnetization in the absence of an external field, indicating that the MnFe₂O₄ NPs are superparamagnetic at 300 K. The MnFe₂O₄ NPs saturation magnetization was 68.2 emu/g (A). The linear fitting of concentration and 1/T₂ showed that the r₂ of PEG-b-PCL-MnFe₂O₄ NMs was 281.7 mM⁻¹ s⁻¹, which was ~4.2 times higher than that of TMAH-MnFe₂O₄ NPs (67.5 mM⁻¹ s⁻¹) (B). Abbreviations: NMs, nanomicelles; NPs, nanoparticles; PEG-b-PCL, polyethylene glycol-block-poly(ε-caprolactone); r₂, transverse relaxivity; T₂, transverse relaxation time; TMAH, tetramethylammonium hydroxide.

Figure 5

MRI of MnFe₂O₄ TMAH-MnFe₂O₄ NPs and PEG-b-PCL-MnFe₂O₄ NMs. Notes: TMAH-MnFe₂O₄ NPs and PEG-b-PCL-MnFe₂O₄ NMs both exhibited a concentration-dependent signal drop in the GRE T₂WI and FSE T₂WI. The signal intensity decreased gradually with the increase of the metal ions concentration, while NMs induced greater hypointensity at an identical concentration compared with NPs. Abbreviations: MRI, magnetic resonance imaging; NMs, nanomicelles; NPs, nanoparticles; PEG-b-PCL, polyethylene glycol-block-poly(ε-caprolactone); TMAH, tetramethylammonium hydroxide; T₂WI, T₂-weighted imaging; GRE, gradient echo; FSE, fast spin echo.

Figure 6

Cytotoxicity analysis. Notes: The RCV of TVECs labeled with CL-PEG-MnFe₂O₄ within 0.9 mM decreased only 7%–9% compared to controls. As the concentration increased, the RCV decreased correspondingly, and a significant decrease of ~19% in viability of TVECs incubated with 4.5 mM CL-PEG-MnFe₂O₄ solution was measured. On the contrary, a notable decrease of ~15% in viability of TVECs incubated with only 0.05 mM MnCl₂ solution was observed. When the TVECs were incubated with MnCl₂ solution as high as 4.5 mM, there were only 36.2% cells that survived. Abbreviations: NMs, nanomicelles; PEG, poly(ethylene glycol); RCV, relative cell viability; TVECs, tumor vascular endothelial cells.

Figure 7

ROS of TVECs labeled with CL-PEG-MnFe₂O₄. Notes: The ROS levels of TVECs labeled with 0.05 (B), 0.23 (C), 0.45 (D), and 0.9 (E) mM CL-PEG-MnFe₂O₄ solutions were very low and depicted an insignificant elevation of ROS level compared with the control (A). However, a 3.18% higher intracellular ROS level was measured in the cells incubated with 4.5 mM CL-PEG-MnFe₂O₄ solution compared with 0.9 mM CL-PEG-MnFe₂O₄ (F). Abbreviations: PEG, poly(ethylene glycol); ROS, reactive oxygen species; TVECs, tumor vascular endothelial cells.

Figure 8

In vitro labeling of TVECs with CL-PEG-MnFe₂O₄. Notes: Prussian blue staining (200×) of control group (A), TVECs labeled with CL-PEG-MnFe₂O₄ (B), PEG-b-PCL-MnFe₂O₄ (C) and the mixture of CL-PEG-MnFe₂O₄ and CL 1555 peptides (D). It showed that the uptake of CL-PEG-MnFe₂O₄ was high, while few PEG-b-PCL-MnFe₂O₄ was engulfed into TVECs. Blocking CD105 with the free CL 1555 peptides also effectively reduced the amount of blue granules in the cytoplasm of TVECs. (E–H) showed the T2-weighted MR images of the four groups of cells. Correspondingly, the cells co-cultured with CL-PEG-MnFe₂O₄ (F) showed a more noticeable signal intensity drop than that with PEG-b-PCL-MnFe₂O₄ (G) and blocking with the free CL 1555 peptides (H). Abbreviations: MR, magnetic resonance; PEG-b-PCL, polyethylene glycol-block-poly(ε-caprolactone); TVECs, tumor vascular endothelial cells.

Figure 9

MRI of subcultured labeled TVECs. Notes: MRI showed that the signal intensity from P₁ to P₄ in T₂WI gradually increased and P₄ had almost equal signal intensity with control group. In T₁WI, the signal intensity from P₁ to P₃ gradually increased and that of P₄ came down to the control. The plot showed that the T₁ relaxation time from P₁ to P₄ decreased firstly and then increased back. Abbreviations: MRI, magnetic resonance imaging; T₁WI, T₁-weighted imaging; T₂WI, T₂-weighted imaging; TVECs, tumor vascular endothelial cells.