VHPKQHR Peptide Modified Ultrasmall Paramagnetic Iron Oxide Nanoparticles Targeting Rheumatoid Arthritis for T1-Weighted Magnetic Resonance Imaging

Abstract

Magnetic resonance imaging (MRI) could be the ideal diagnostic modality for early rheumatoid arthritis (RA). Vascular cell adhesion molecule-1 (VCAM-1) is highly expressed in synovial locations in patients with RA, which could be a potential target protein for RA diagnosis. The peptide VHPKQHR (VHP) has a high affinity to VCAM-1. To make the contrast agent to target RA at an early stage, we used VHP and ultrasmall paramagnetic iron oxide (USPIO) to synthesize UVHP (U stands for USPIO) through a chemical reaction with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide. The size of UVHP was 6.7 nm; the potential was −27.7 mV, and the r2/r1 value was 1.73. Cytotoxicity assay exhibited that the cell survival rate was higher than 80% at even high concentrations of UVHP (Fe concentration 200 µg/mL), which showed the UVHP has low toxicity. Compared with no TNF-α stimulation, VCAM-1 expression was increased nearly 3-fold when mouse aortic endothelial cells (MAECs) were stimulated with 50 ng/mL TNF-α; cellular Fe uptake was increased very significantly with increasing UVHP concentration under TNF-α treatment; cellular Fe content was 17 times higher under UVHP with Fe concentration 200 µg/mL treating MAECs. These results indicate that UVHP can target overexpression of VCAM-1 at the cellular level. RA mice models were constructed with adjuvant-induced arthritis. In vivo MRI and biodistribution results show that the signal intensity of knee joints was increased significantly and Fe accumulation in RA model mice compared with normal wild-type mice after injecting UVHP 24 h. These results suggest that we have synthesized a simple, low-cost, and less toxic contrast agent UVHP, which targeted VCAM-1 for early-stage RA diagnosis and generates high contrast in T1-weighted MRI.

T1-weighted MRI of targeting atherosclerotic plaque based on CD40 expression on engulfed USPIO’s cell surface

“…Synthesis of USPIO can be seen in our previous experimental methods [13]. USPIO is synthesized by the following steps: 8 mM anhydrous FeCl 3 was dissolved in 40 ml of diglycol.…”

“…The resulting black product was dissolved in water and lyophilized to obtain USPIO powder for the next step. USPIO shape with transmission electron microscopy (TEM) observation, zeta potential and dynamic light scattering meas-urements, specific method please refer to previous articles published by our laboratory [13].…”

“…Then the digested solution was made up to 10 ml with deionized water, and then detected by ICP-OES (Inductively Coupled Plasma Optical Emission Spectrometer, Thermo E. IRIS Duo) [12] MAECs and EPCs not treated with USPIO were used as controls. The iron content is measured by ICP-OES, for the conversion of the relationship between iron content and USPIO, please refer to our previously published article [13].…”

“…The USPIO were synthesized in alignment with our previous experimental methods [13]. The explicit synthesis method is comprehensively described in the Material and Methods section 2.2.…”

“…USPIO (<5 nm) have been used as T1 contrast agents for MR imaging applications [12]. We previously used this USPIO with peptide linkage to achieve targeting inflammation sites with significant difference in T1-weighted MRI [13], which raise this question-could phagocytosed USPIO's EPCs (USPIO-EPCs) and mouse aortic endothelial cells (USPIO-MAECs) perform T1-weighted MRI? This query warrants worth further research.…”