Hypersensitive MR angiography based on interlocking stratagem for diagnosis of cardiac-cerebral vascular diseases

Magnetic resonance (MR) angiography is one of the main diagnostic approaches for cardiac-cerebral vascular diseases. Nevertheless, the non-contrast-enhanced MR angiography suffers from its intrinsic problems derived from the blood flow-dependency, while the clinical Gd-chelating contrast agents are limited by their rapid vascular extravasation. Herein, we report a hypersensitive MR angiography strategy based on interlocking stratagem of zwitterionic Gd-chelate contrast agents (PAA-Gd). The longitudinal molar relaxivity of PAA-Gd was 4.6-times higher than that of individual Gd-chelates as well as appropriate blood half-life (73.8 min) and low immunogenicity, enabling sophisticated micro-vessels angiography with a resolution at the order of hundred micrometers. A series of animal models of cardiac-cerebrovascular diseases have been built for imaging studies on a 7.0 T MRI scanner, while the clinical translation potential of PAA-Gd has been evaluated on swine on a 3.0 T clinical MRI scanner. The current studies offer a promising strategy for precise diagnosis of vascular diseases.


Supplementary Note 1. Synthesis and Characterization of PAA-Gd
The synthetic route of zwitterionic MCP contrast agent PAA-Gd is illustrated in Figure S1.DTPA was covalently linked to PAA through the DET linker via amidation reaction, which was catalyzed by DMTMM.The PAA-Gd contrast agent was obtained through the chelating reaction between residual carboxyl groups of DTPA and Gd 3+ ions.
The 1 H NMR spectra of PAA, PAA-DET and PAA-DTPA in each synthesis step According to the integrated peaks areas, ~92.0% of carboxyl groups in PAA were convert to amino groups after conjugating with DET, and ca.86.0% of carboxyl groups were finally conjugated with DTPA.The amount of Gd 3+ chelated by PAA-Gd was estimated through thermo gravimetric analysis (TGA), and the result suggests that almost all of DTPA groups (~99.7%)chelate Gd 3+ , thus the Gd 3+ content of the polymeric contrast agent is ca.1.2 mmol/g MCP.
In addition, the polydispersity indices (PDI) of PAANa and PAA-DTPA measured by an aqueous gel-permeation chromatography (GPC) system (GPC Waters 1515) calibrated with poly(ethylene glycol) standards were 1.20 and 1.32, respectively, indicating that the samples were well dispersed and no inter-or intra-molecular crosslinking occurred during the synthesis process.
The impact of the above synthesis process on the properties of the polymer was further investigated with dynamic light scattering (DLS) carried out at a Nano Zetasizer (Malvern).The results in Figure 1c revealed that the hydrodynamic diameter (dH) of PAA molecules is reasonably increased from 4.0 nm to 4.9 nm after conjugation of DET linkers, and continue to increase to 5.1 nm after PAA-DTPA was synthesized.After Gd 3+ chelation, the dH of polymers eventually grew to 7.7 nm, implying that the PAA-Gd molecule is successfully constructed.Besides, the DLS profile remains nearly unchanged through the conjugation and coordination reaction except for the shift owing to the increased size of polymers, suggesting that the conjugation reaction took place in a controlled manner, which did not lead to unwanted aggregates.
In addition, the impact of the conjugation and coordination reaction on the electrophoretic mobility of the polymers were also investigated (Figure 1d).The electrophoretic mobility can be determined by the Henry Equation: where UE is the electrophoretic mobility, ε is the dielectric constant, z is the zeta potential, f(Ka) is Henry's function with value of 1.5 in PBS buffer, and η is the viscosity.In physiological PBS buffer (pH 7.4), the electrophoretic mobility of PAA polymers significantly increased from -1.26 m 2 V -1 s -1 10 -8 to 0.64 m 2 V -1 s -1 10 -8 during the DET conjugation, which suggested that the carboxyl groups in the side chains of PAA have been convert to amino groups.Thereafter, through the connection of DTPA, the electrophoretic mobility of polymers decreased to -1.37 m 2 V -1 s -1 10 -8 due to the multiple carboxyl groups in the DTPA molecules.At last, through being coordinated with Gd 3+ , the electrophoretic mobility of PAA-Gd molecule was re-increased to -0.24 m 2 V -1 s -1 10 -8 owing to the positive charge carried by the Gd 3+ ions, which is almost neutral in physiological conditions.This variation of electrophoretic mobility implied that after the above-mentioned multi-step reaction, the zwitterionic PAA-Gd molecules were successfully synthesized.

Supplementary Note 2. Calculation of the Elimination Half-life of Contrast Agent Through Two-compartment Model
In the two-compartment model, the decline of drug concentration can be divided into two distinct phases, i.e., the distribution phase with initial rapid decline in serum drug concentration, and the elimination phase with slow decline in drug concentration.
The clearance of drugs can be mathematically described as a bi-exponential function: Where t is the time since the administration of drug; C(t) is the drug concentration; A and B are coefficients that describe the exponential functions of distribution phase and elimination phase; α and β are exponents that describe the shape of the curve for distribution phase and elimination phase.
After the distribution of the drug achieve an equilibrium, the above function can be simplified as: Therefore, the elimination half-life of the drug can be expressed as: Accordingly, through data fitting, the elimination half-life of the PAA-Gd and Gd-DTPA contrast agent can be calculated as 73.8 and 11.4 min, respectively.

Supplementary Figures
Figure S1 The synthetic route of zwitterionic PAA-Gd contrast agent.

(
carried out on a 400 MHz Nuclear Magnetic Resonance Spectrometer (AVANCE III)) are displayed in the Figure S2-S4 (analyzed by MestReNova software (9.0.1.13254)).The proton signals of methylene and methylidyne on the backbone of PAA (a & b) appeared at 1.40 and 2.02 ppm, respectively.After conjugation with diethylenetriamine (DET) and DTPA in turn, the proton signals of the methylene of DET (c, d, e and f), and the proton signals of the methylene of DTPA (g, h, i and j) appeared within the range of 2.50-4.10ppm.The proton signals of the backbone (a & b) moved slightly to a higher magnetic field, indicating that DET and DTPA were successfully combined with the main chain of PAA.

Figure
Figure S2 1 H NMR Spectra (D2O) of PAANa with the numbers of hydrogen atoms determined by integration of the peak areas.

Figure S3 1 H
Figure S3 1 H NMR Spectra (D2O) of PAA-DET with the numbers of hydrogen atoms determined by integration of the peak areas.(Approximately 49 of the 54 carboxyl groups on the side chain of PAA molecule are attached to DET).

Figure
Figure S4 1 H NMR Spectra (D2O) of PAA-DTPA with the numbers of hydrogen atoms determined by integration of the peak areas.(Approximately 42 of the 49 -NH2 of PAA-DET molecule were further coupled to DTPA).

Figure S5
Figure S5 Temporal evolution of the average intravascular relative signal intensity before and at different time points after intravenous injection of PAA-Gd or Gd-DTPA, respectively (n = 3, data were plotted as mean ± standard deviation).

Figure S6
Figure S6 Temporal evolution of the average liver parenchyma relative signal intensity before and at different time points after intravenous injection of PAA-Gd or Gd-DTPA, respectively (n = 3, data were plotted as mean ± standard deviation).

Figure S7
Figure S7 TOF MR angiography of rat head obtained before PAA-Gd injection.The embedded scale bar corresponded to 5 mm.Triplicates were performed independently with similar results.

Figure
Figure S8 PAA-Gd-enhanced 3D MR angiography of the rat head after ischemic stroke, in which the left carotid artery and the compensatory angiogenesis surrounding the blocked right carotid artery were indicated by the red arrow and yellow arrows, respectively.The embedded scale bar corresponded to 5 mm.Triplicates were performed independently with similar results.

Figure
Figure S9 PAA-Gd-enhanced 3D MR angiography of the mouse head acquired through using conventional head coil.The embedded scale bar corresponded to 2 mm.Triplicates were performed independently with similar results.

Figure
Figure S10 the transmission electron microscope (left) image, size distribution profiles (middle), and DLS result (right) of PEGylated NaGdF4 nanoparticles.The embedded scale bar corresponded to 30 nm.At least five repetitions were performed independently with similar results.Transmission electron microscope image was conducted on a transmission electron microscope (JEOL, JEM-2100).

Figure S11
Figure S11 H&E staining of tissue slices of major organs from mice treated with different agents.The embedded scale bar corresponded to 50 μm.Triplicates were performed independently with similar results.

Figure S12
Figure S12 CPNIII staining of tissue slices of major organs from mice treated with different agents.The embedded scale bar corresponded to 50 μm.Triplicates were performed independently with similar results.

Figure S13
Figure S13 Cell viability assay of PAA-Gd and Gd-DTPA contrast agent (n = 3, data were plotted as mean ± standard deviation).

Figure S14
Figure S14Hemolysis rates of PAA-Gd contrast agent at different Gd 3+ concentrations (n = 3, data were plotted as mean ± standard deviation).

Figure S15
Figure S15 H&E staining of kidney tissues from the representative rats in each group.The embedded scale bar corresponds to 200 μm.Triplicates were performed independently with similar results.

Figure S16
Figure S16 H&E staining of skin tissues from the representative rats in each group.The embedded scale bar corresponds to 200 μm.Triplicates were performed independently with similar results.

Figure
Figure S17 TOF MR angiography of the FeCl3-induced carotid arterial thrombosis mouse model.

Figure S18
Figure S18 3D TRICKS of swine acquired pre-and post-injection of Gd-DTPA at the dose of 0.1 mmol per kg weight and PAA-Gd at the dose of 0.1 mmol/kg or 0.03 mmol/kg, respectively.

Figure S19
Figure S19 Temporal evolution of the average intravascular MRI signal intensity before and at different time points after intravenous injection of PAA-Gd (0.03 mmol/kg) or Gd-DTPA (0.1 mmol/kg), respectively (n = 3, data were plotted as mean ± standard deviation).

Figure S20
Figure S20 The vascular signal-to-background ratio between five different blood vessels and their surrounding normal tissues (n = 5) of swine after injecting with PAA-Gd or Gd-DTPA, respectively, together with their statistical difference.Statistical significance was determined by two-sided unpaired T-test, p = 0.02 (**p < 0.01).Data were plotted as mean ± standard deviation.

Figure S21
Figure S21 Temporal evolution of the average intravascular T1 signal intensity before and at different time points after intravenous injection of PAA-Gd or Gd-DTPA, respectively (n = 3, data were plotted as mean ± standard deviation).

Figure S22
Figure S22 Delayed 3D BRAVO T1-weighted MRI obtained at different time points post-injection of PAA-Gd with 0.03 mmol/kg.

Figure S23
Figure S23 Temporal evolution of the T1 relative signal intensity of the bladder region (n = 3).

Figure S24
Figure S24 Delayed 3D BRAVO T1-weighted MRI obtained at different time points post-injection of Gd-DTPA with 0.1 mmol/kg.

Figure
Figure S25 3D TOF MR angiography of swine.

Figure S26
Figure S26 3D PC MR angiography of swine.

Figure S27
Figure S27 Fluctuations in the rectal temperature and the increase of body weight of young swine after PAA-Gd administration.

Figure S28
Figure S28 Blood biochemical test results of young swine treated with the PAA-Gd contrast agents.

Figure S29
Figure S29 Routine blood test results of young swine treated with the PAA-Gd contrast agents.

Figure S30
Figure S30 H&E staining of tissue slices from major organs of young swine treated with the PAA-Gd at 33 d post-injection.The embedded scale bar corresponded to 500 μm.Triplicates were performed independently with similar results.

Figure
Figure S31 CPN III staining of tissue slices of major organs from swine injected with PAA-Gd (Gd 0.1 mmol/kg).The embedded scale bar corresponded to 50 μm.Triplicates were performed independently with similar results.

Figure S32
Figure S32The digital photograph of practical equipment of the large-scaled PAA-Gd synthesis system (left) and the standardly packaged PAA-Gd powder and injection for the potential clinical use (right).