Preparation and characterization of Gd-chelated PEG-TCPP nanoparticles
The GPT nanoparticles were synthesized by complexing TCPP with 8-arm-PEG4k-NH2 and induced by dimethyl sulfoxide (DMSO) at room temperature under argon gas protection via a previously reported method . Then, the introduced Gd3+ was chelated to the center of TCPP to endow the GPT NPs with paramagnetic property, which could act as the contrast agents for T1-weighted MRA. The as-synthesized GPT NPs were further purified by PD-10 columns several times to decrease particle aggregation and residual Gd3+ ions in the nanoparticle suspensions. Transmission electron microscopy (TEM) images indicate that as-prepared GPT NPs have well-fabricated spherical structure (Fig. 1a and Additional file 1: Fig. S1) with the average size of 30 nm. X-ray energy dispersive spectroscopy (EDS) shown in Fig. 1b verifies the existence of Gd elements in GPT NPs. The average hydrodynamic diameter of GPT NPs suspended in water is 35 nm as determined by dynamic light scattering (DLS) (Fig. 1c). The chelated Gd in GPT NPs is validated by the presence of the characteristic peaks corresponding to Gd 4d (142 eV) in the X-ray photoelectron spectroscopy (XPS) spectra (Fig. 1d). The presence of Gd 4d peaks indicates the oxidation state of the chelated Gd, confirming the synthesis approach is mild meanwhile the physicochemical environment for Gd coordination is still well preserved. It is noted that the XPS analysis verifies GPT NPs are composed of C, O and Gd, as evidenced by the characteristic peaks corresponding to C 1s (284 eV), O 1s (532 eV), and Gd 4d (142 eV). The results reveal the presence of the carboxyl group on the surface of GPT NPs, offering good physiological stability. Fourier Transform Infrared (FTIR) spectroscopy was applied to characterize the formation of GPT NPs. With the chelation of TCPP and presence of PEG containing groups, there were lots of surface amines and carboxyl groups on the surface of GPT NPs such as O–H (3432 cm− 1), N–H (2886 cm− 1), C=C (1700 cm− 1), and C–O (1112 cm− 1) (Additional file 1: Fig. S2). In addition, fluorescence spectroscopy analysis also confirms the presence of TCPP in GPT NPs (Additional file 1: Fig. S3). Free Gd3+ ions can hardly leak from GPT NPs, because TCPP is an excellent chelator for metal ions. These results revealed that only a negligible number of free Gd3+ ions were released from GPT NPs (Additional file 1: Fig. S4).
In vitro T1-weighted MR imaging performance
To estimate the performance of GPT NPs as T1–weighted contrast agents, the r1 value was determined by taking the slope of the linear plot of 1/T1 versus Gd concentration. GPT NPs exhibit the contrast enhancement in T1–weighted MR images (Fig. 2a), with r1 value of 35.76 mM− 1 s− 1 at 3.0 T, indicating a significant increase as compared to that of commercial Gd contrast agents Omniscan (5.41 mM− 1 s− 1) (Fig. 2b, c). Owing to the high content of chelated Gd3+ with the TCPP and the direct interactions between Gd3+ and hydrogen protons, the r1 value of GPT NPs is much higher than that of clinical used Gd-based contrast agents such as Omniscan (3.3 mM− 1 s− 1) , Magnevist (4.1 mM− 1 s− 1) , ProHance (4.3 mM− 1 s− 1) , and Gadovist (4.34 mM− 1 s− 1) . These results further demonstrate that GPT NPs have significant potential for excellent T1–contrast agents for MRA contrast enhancement.
MR performance in vivo
To investigate the performance of GPT NPs-enhanced MRA, biocompatible GPT NPs were intravenously (i.v.) injected into Sprague-Dawley rats for vascular imaging under a 3.0 T clinical MRI scanner. Immediately, the common cartied artery, subclavian artery, heart, aorta, and common iliac artery can be clearly differentiated by GPT NPs-enhanced MRA, whereas Omniscan-enhanced MRA images exhibit weak contrast enhancement post-injection (Fig. 3a, b). Remarkably, as revealed by the MR-signal intensities images, the signal intensity value of aorta by GPT NPs with a prolonged time window is much higher than that of using Omniscan, demonstrating the superior contrast effect of GPT NPs for long scan time and arterial vascular anatomy. Moreover, as revealed by the prolonging of duration after the intravenous administration of MRA images, GPT NPs were retained in blood with prolonged vascular enhancement (360 min) as compared to Omniscan (60 min), because of the long blood half-life of GPT NPs (Fig. 3b, c). Although the dosage of GPT NPs was 0.1 mmol kg− 1 of Gd as the same as the commercial T1 contrast agent Omniscan, arterial vessels of GPT NPs were substantially brightened, while contrast enhancement in the surrounding tissue was negligible. GPT NPs allowed rapid imaging of purely arterial image and the minimization of overlap with enhancing veins and tissue. On the contrary, a comparative study using Omniscan exhibited weak contrast in the whole-body vessels, which rendered it difficult to receive detailed vascular diagnostic information by a single injection. The MRA performance of GPT NPs showed superior vascular imaging quality and acceptance of high-spatial-resolution. The blood circulation curve illustrated that the pharmacokinetics of GPT NPs followed a two-compartment model with the half-time (T1/2) of 6.007 h (Additional file 1: Fig. S5). The negligible free Gd3+ leakage from GPT NPs by measuring the Gd concentration in blood of health rats at varied time intervals (Additional file 1: Fig. S6). These results demonstrated that GPT NPs were biocompatible with low cytotoxicity.
Inspired by the desirable rats’ vascular performance, GPT NPs-enhanced MRA images were further conducted on larger animals (New Zealand rabbit model). GPT NPs exhibited excellent MRA performance in the visualization of upper-extremity vessels, including the common cartied artery, vertebral artery, subclavian artery, aorta, and heart. GPT NPs also enable clear visualization of lower-extremity vessels, including the common iliac artery, external iliac artery, deep femoral artery, and femoral artery. Under identical experimental conditions, MRA performance GPT NPs was superior to Omniscan, possibly due to its long-circulating and excellent signal-to-noise (Fig. 4a).
Heart is one of the most important organs of circulation that pumps blood to the vascular system. Cardiovascular magnetic resonance imaging has emerged as an indispensable non-invasively method to discern abnormal cardiovascular disease and cardiomyopathies . However, the technique has been limited due to difficulties generated by standard extracellular contrast agents resulting in short circulating time and rapid background signal, which hampers the high-resolution MRA. Thus, the development of novel contrast agents that provide prolonged vascular enhancement and highly efficient MRA is very meaningful for clinical application. Herein, cardiovascular magnetic resonance imaging was further examined with intravenous injection of GPT NPs. Without the GPT NPs-assisted MRA, the cardiac structure is hardly observable (Fig. 4b upper). After the injection of GPT NPs, there is an immediate increase in signal intensity of the heart, including the brachiocephalic trunk, aortic arch, ascending aorta, aortic sinus, right ventricle, left ventricle, and interventricular septum (Fig. 4b lower), demonstrating the superior contrast effect of GPT NPs for assessing the cardiac vascular anatomy.
Motivated by the superior relaxivity and high SNR, biocompatible GPT NPs were injected (i.v.) into the mice bearing the subcutaneous PANC-1 pancreatic tumors and performed T1-weighted MRI. Omniscan with the equivalent concentration of Gd were used as controls. MR images at different time points before and after administration of these contrast agents are shown in Fig. S7. For a fair comparison, the two groups’ MRI signals of tumor sites were compared at given time points. There were significant enhancement signals in the GPT NPs group, compared with the Omniscan treated mice. It is obvious that the T1 contrast in the tumor sites is the strongest at 30 min for GPT NPs or 5 min post-injection for Omniscan, respectively (Additional file 1: Fig. S8). In addition, the MRI signal of GPT NPs at 4 h post-injection is still much stronger than that of the Omniscan at 5 min post-injection due to the long-time blood circulation time and super high r1 value.
Biocompatibility and biosafety
An ideal MR contrast agents need to exhibit biocompatibility and biosafety. In general, free Gd3+ ions are considered to be cytotoxic. Therefore, it is necessary to investigate the cytotoxic effect of GPT NPs in the physiological environment. The cytotoxicity of GPT NPs and Omniscan in vitro was determined by the cell counting kit-8 (CCK-8) assay on pancreatic ductal epithelial cells (PDEC) and human pancreatic cancer PANC-1 cells. These cells were incubated with GPT NPs and Omniscan at elevated concentrations (0, 0.125, 0.25, 0.5, 1 mM) for 24 h and no obvious cytotoxicity was observed even at a concentration as high as 1 mM, demonstrating the low cytotoxicity and excellent biocompatibility of GPT NPs (Additional file 1: Figs. S9a and S10a). Then PDEC cells were incubated with GPT NPs time as long as 48 h. As expected, with the prolonged time, both GPT NPs and Omniscan showed negligible cytotoxicity with over 90% cell viability (Additional file 1: Fig. S9b and Sb). The relatively lower cytotoxicity of GPT NPs can be ascribed from the stabilization of Gd-chelates, which Gd3+ ion is very hard to be released from the nanoparticles.
To further evaluate the long-term in vivo toxicity and biocompatibility of GPT NPs, body-weight changes, hematological assessments, and H&E staining were systematically performed. Healthy BALB/c mice were i.v. injection of GPT NPs at elevated doses (0, 5, 10, 20 mg kg− 1) and then fed for 1-month period. No significant difference in body weight was observed compared to the control group, demonstrating the low toxicity of GPT NPs under different concentrations (Additional file 1: Fig. S11). The blood biochemistry of mice, including liver and kidney function after the i.v. administration of GPT NPs, further confirmed the biosafety of GPT NPs. Additional file 1: Fig. S12 shows no obvious hepatic toxicity (by measuring the serum levels of ALT, AST, and ALP) and Additional file 1: Fig. S13 shows no obvious kidney toxicity (by measuring the serum levels of BUN and CR) among the control group and the treatment groups. For hematological analyses, including the indexes of white blood cells analysis (Additional file 1: Fig. S14), platelets analysis (Additional file 1: Fig. S15), hemoglobin and red blood cells analysis (Additional file 1: Fig. S16, S17), all measured indicators appeared to be normal compared to those in the control group. Histological analyses of the main organs (heart, liver, spleen, lung, and kidney) in all groups were performed by hematoxylin and eosin (H&E) staining, and no significant tissue abnormalities or severe inflammation were detected in these tissue (Additional file 1: Fig. S18). All aforementioned biocompatibility analysis results elucidates the low toxicity of GPT NPs under administration dose. The GPT NPs are intrinsically featured with good biocompatibility for potential clinical translation, especially for further T1-weighted MRA imaging.