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. 2024 Feb 16;16(8):9702–9712. doi: 10.1021/acsami.3c16391

Amorphous Albumin Gadolinium-Based Nanoparticles for Ultrahigh-Resolution Magnetic Resonance Angiography

Chenchen Liu †,‡,⊥, Xiaoming Liu §,∥, Zhihao Wei †,‡, Zong Chang ⊥, Yaowei Bai §,∥, Pei Zeng †,‡, Qi Cao †,‡, Changjun Tie #, Ziqiao Lei §,∥, Peng Sun , Huageng Liang †,‡,*, Qinchao Sun ⊥,*, Xiaoping Zhang †,‡,*
PMCID: PMC10911108  PMID: 38363797

Abstract

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Magnetic resonance angiography (MRA) contrast agents are extensively utilized in clinical practice due to their capability of improving the image resolution and sensitivity. However, the clinically approved MRA contrast agents have the disadvantages of a limited acquisition time window and high dose administration for effective imaging. Herein, albumin-coated gadolinium-based nanoparticles (BSA-Gd) were meticulously developed for in vivo ultrahigh-resolution MRA. Compared to Gd-DTPA, BSA-Gd exhibits a significantly higher longitudinal relaxivity (r1 = 76.7 mM–1 s–1), nearly 16-fold greater than that of Gd-DTPA, and an extended blood circulation time (t1/2 = 40 min), enabling a dramatically enhanced high-resolution imaging of microvessels (sub-200 μm) and low dose imaging (about 1/16 that of Gd-DTPA). Furthermore, the clinically significant fine vessels were successfully mapped in large mammals, including a circle of Willis, kidney and liver vascular branches, tumor vessels, and differentiated arteries from veins using dynamic contrast-enhanced MRA BSA-Gd, and have superior imaging capability and biocompatibility, and their clinical applications hold substantial promise.

Keywords: magnetic resonance angiography, ultrahigh-resolution, amorphous nanoparticles, albumin, gadolinium

Introductions

High-resolution imaging of the microvessels is critical for monitoring and treating a variety of diseases, designing the surgical route, and planning preoperative embolization.1,2 Magnetic resonance angiography (MRA) is one of the most powerful tools for vascular imaging in a radiation-free manner.3−5 However, the commonly used gadolinium-based contrast agents for high-resolution MRA present limitations in terms of the effective time window and longitudinal relaxivity (r1).6 Recording vessels with a diameter of about a few hundred micrometers is a formidable task even with the most advanced MRA techniques,7 for example, complete recognition of the clinically significant circle of Willis and tumor vessels.8−10 To reduce the scanning time and administration dose, another area of concern is the realization of one-time, wide-area imaging.11 Identification of arteries and veins through dynamic contrast-enhanced MRA is considered equally critical for the diagnosis of diseases including arteriovenous malformations.12 Moreover, imaging certain specific vessels still poses great challenges. For instance, kidney vasculature imaging is dramatically compromised as most of the commercial contrast agents are excreted through kidneys.7,13

For decades, inorganic, organic, and hybrid nanomaterials have been designed to obtain an MRA contrast agent with high r1.14−17 The albumin-coated nanomaterials are considered one of the most promising MRA contrast agents as they have high biocompatibility, colloidal stability, and an extended blood half-life for drug delivery.18,19 The preparation of BSA-coated Gd2O3 nanomaterials (albumin-coated gadolinium-based nanoparticles) was reported by mixing BSA and Gd3+ under strong bases.20,21 The r1 of the reported BSA-coated Gd2O3 was around 12 mM–1 s–1, which was about 3-fold that of Gd-DTPA (a Food and Drug Administration-approved contrast agent). Although these contrast agents are promising, they still possess certain undesirable key parameters, such as the relatively low value of r1. Contrast agents with higher r1 can either provide greater tissue enhancement and thus facilitate better detection of smaller lesions22 or lower the administration dose for achieving a similar image contrast, which may reduce the risk of Gd-induced toxicity.23,24 Furthermore, for achieving high-resolution MRA, in addition to applying a high magnetic field, prolonged imaging time under a relatively low field like 3T would be a more practical approach. Therefore, the blood circulation time of contrast agents is crucial in realizing the high-resolution MRA. However, the rapid transient metabolism in the vasculature and nonspecific tissue distribution patterns of the commercial gadolinium-based contrast agents make the long-term MRA scan infeasible.6,25 More importantly, their efficacy should be evaluated in large mammals to verify their clinical translation potential.

Herein, we present an extraordinary MRA contrast agent, albumin-coated gadolinium-based nanoparticles (BSA-Gd), with a dramatically enhanced T1 signal. The performance of BSA-Gd in rats, rabbits, and beagles was fully confirmed via MRA; for instance, vessels of sub-200 μm and even finer vascular branches in the rat liver and kidneys were recorded after the bolus intravenous administration of BSA-Gd. Accordingly, extensive as well as disorderly trophoblastic vessels of implanted tumors in the muscle and kidney can also be clearly labeled in vivo via 3T magnetic resonance (MR). We successfully performed a one-time whole-body MRA imaging in beagles and observed the major vessels, particularly the spinal vessels. Consistent with the theory, the carotid artery, jugular vein, aorta, inferior vena cava, and subclavian vein of the rat were clearly displayed at a very low dose of Gd, about 1/16 of the clinically recommended dose. These significant results suggest that BSA-Gd is highly valuable in the diagnosis and treatment of vascular lesions or vascular-related diseases. Characteristics such as the simplicity, the low cost of the synthesis process, and the excellent stability of BSA-Gd provide an opportunity for industrial scale production.

Materials and Methods

Materials

Gadolinium(III) chloride (GdCl3, 99.95%) was procured from Shanghai Aladdin Biochemical Technology Co., Ltd. BSA (chromatographically purified, ≥98% purity) was purchased from Aldrich. The commercially used MRA contrast Gd-DTPA (20 mL: 9.38 g) was purchased from Beijing Beilu Pharmaceutical Co., Ltd. All of the reagents were used as received without further purification.

Synthesis of Albumin-Coated Gadolinium-Based Nanoparticles

GdCl3 (11.7 mg) was dissolved in 2 mL of distilled water, and 2 mL of NaOH (pH about 12) was added dropwise and mixed by vortexing. The produced precipitation was collected by centrifugation at 9000g for 3 min and washed thrice. Then, the precipitation was resuspended in 4 mL of distilled water, and a solution of BSA (30 mg/mL, 10 mL) was added dropwise and mixed by vortexing. The mixture was stirred at room temperature for 2 h and then centrifuged at 9000g for 3 min to remove large-size particles. Finally, the supernatant was concentrated and purified using a 100 kDa molecular weight cutoff (MWCO) ultrafiltration tube (15 mL-capacity; Millipore). The product was freeze-dried and stored in a refrigerator at 4 °C until further use.

Measurements of r1

To assess the T1 value of BSA-Gd, the samples in 0.01 M phosphate buffered saline (PBS) were scanned using a 2D fast spin echo sequence with the following parameters: repetition time (TR) = 5000 ms; echo time (TE) = 13.3 ms; flip angle = 120°; number of averages = 1; slice thickness = 5 mm; and echo train length = 10. For the r1 calculation, the equation r1 = (1/T1 – 1/T0)/[Gd] was utilized. [Gd] represents the concentration of Gd in BSA-Gd (in mM). Here, 1/T1 (in s–1) and 1/T0 (in s–1) are the longitudinal relaxation rates with or without the contrast agent, respectively.

Magnetic Resonance Imaging of Resolution Phantoms

The MRA spatial resolution was investigated at different scanning parameters by using a phantom filled with distilled water. A gradient recalled echo (GRE) scanning sequence was utilized with the below-mentioned parameters to acquire the images of the phantoms under 3T MR: repetition time = 4.2/5.2/5.7/6.7/10.9/15.8 ms; echo time = 1.6/1.9/2.1/2.4/3.2/3.9 ms; flip angle = 20.0°; number of averages = 2; and slice thickness = 0.5 mm.

Cell Lines

The human normal liver cell line (LO2) and human umbilical vein endothelial cell line (HUVEC) were a kind gift from Jiao Xie (Huazhong University of Science and Technology, China). HK-2 (CRL-2190), Raw264.7 (TIB-71), and C6 (CCL-107) cells were obtained from the American Type Culture Collection. LO2 cells were maintained in RPMI1640 medium in a 37 °C incubator with 5% CO2. HK-2, HUVEC, Raw264.7, and C6 cells were cultivated in Dulbecco’s modified Eagle medium. All media were supplemented with 10% fetal bovine serum and 1% streptomycin/penicillin.

Animals

Sprague–Dawley rats were obtained from HuBei Biont Bio-Technology CO. Ltd. New Zealand rabbits (weight 2.5 kg) and beagles (weight 10 kg) were purchased from WuHan WanQianJiaXing Bio-Technology Co. Ltd. All experiments on animals were conducted following the guidelines of the National Institute of Health. The experimental procedures were approved by the Institutional Animal Care and Use Committee of the Huazhong University of Science and Technology. Prior to the operation, rats were anesthetized with 2.5% isoflurane in oxygen, and rabbits and dogs were anesthetized with 10% pentobarbital sodium.

Tumor Model

C6 cells were cultured in a T75 flask and collected on reaching a cell density of 80%. The cells were resuspended in PBS and inoculated into the hind limb muscles (5 * 106 cells/each) or kidney (1 * 106 cells/each) of rats. The rats were randomly categorized into different groups 10–14 days after surgery to receive MR scanning.

Contrast Agent Injection for MRA

After the animal was anesthetized, the indwelling needle (24 gauge) was inserted through the tail vein (for rats), ear vein (for rabbits), or forelimb vein (for beagles). Contrast agents were bolus injected when the animals and coils reached the scanning position. Unless otherwise specified, all in vivo imaging experiments were conducted immediately after administration.

Statistical Analysis

All data analysis and plotting were finished via GraphPad Prism 8 software unless otherwise noted. Data were presented as the mean ± standard deviation. The results were analyzed by the Student’s t test between two groups. P < 0.05 was considered significant.

Results and Discussion

Characterization of BSA-Gd Nanoparticles

Amorphous BSA-Gd nanoparticles were prepared as depicted in Figure 1A with modifications to the earlier reported approaches. Briefly, a precipitate was obtained by mixing a solution of GdCl3 and NaOH solution with a pH of about 12. The BSA coating process was done by incubating a solution of BSA with the collected precipitation for 2 h at room temperature. A clear stock solution of BSA-Gd was obtained by removing the insoluble precipitation under centrifugation. The key improvement in the present method is the BSA coating process under a mild condition instead of the denaturing condition of the reported methods. Circular dichroism (CD) spectroscopy was performed for BSA, BSA-Gd, and BSA-Gdx (prepared in a traditional manner) from 200 to 250 nm to investigate the corresponding geometry variation. We may find that the secondary structure of BSA in BSA-Gdx nanoparticles was dramatically destroyed as the CD signal was very low in comparison to that of BSA. The CD spectra of BSA-Gd and BSA were in similar shape and intensity with two characteristic maxima around 209 and 220 nm, indicating that the α-helix structure of BSA in BSA-Gd was well reserved. The hydrodynamic diameter of the prepared BSA-Gd nanoparticle was around 30 nm (Figure 1B), in line with the transmission electron microscopy (TEM) image as shown in Figure 1C. Further, Figure S1 illustrates that BSA-Gdx prepared by traditional methods under strong alkaline conditions has a crystal structure, while BSA-Gd was an amorphous nanoparticle. A negatively charged surface was found with a ζ-potential of about −10 mV, which may be due to the surface-coated BSA. Figure S4 shows that BSA-Gd can exist stably for a long time without aggregation or precipitation in weak acid, alkaline, or different media under environment or physiological temperatures. The X-ray diffraction (XRD) pattern of the precipitation before being coated by BSA exhibits a few broad bands, as shown in Figure 1E, which reasonably overlapped the XRD peaks of Gd2O3. Such characteristic bands were absent for the BSA-Gd, which indicated that the prepared BSA-Gd was in a highly amorphous state. The X-ray photoelectron spectroscopy (XPS) spectrum of Gd(4d) can be divided into two parts with central binding energies of 141.2 and 143.5 eV, which were attributed to Gd(OH)3 and Gd2O3, respectively (Figure S3).

Figure 1.

Figure 1

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Preparation and characterization of BSA-Gd. (A) Preparation procedures of BSA-Gd nanoparticles and the BSA-Gdx (reported). (B) The average size (red column) and ζ-potential (blue column) of BSA-Gd were estimated by dynamic light scattering measurements (repeated five times independently). (C) TEM image of BSA-Gd and the picture of a BSA-Gd solution (inset). (D) CD spectra of BSA, BSA-Gdx, and BSA-Gd. (E) XRD pattern of BSA, BSA-Gd, Gd precipitation, and Gd2O3, JCPDS reference card number 96-101-1289. (F,G) T1, the longitudinal proton relaxation time as a function of the Gd concentration of BSA-Gd and Gd-DTPA, respectively, was measured by 3T MR at 25 °C; the slope represents the relaxivity (r1), n = 1. (H) T1-enhanced contrast images of BSA-Gd and Gd-DTPA at different Gd concentrations taken via 3T MR. (I) In vivo bloodstream MR signal for BSA-Gd and Gd-DTPA as a function of time. The intensity of the MR signal was obtained from the jugular vein (n = 5 for both groups). (J) Assessment of the MRA spatial resolution at different scanning times by Phantom. The images were acquired via 3T MR with a GRE sequence. (K) Scheme for the in vivo T1-enhanced MRA via BSA-Gd nanoparticles in the experimental rat.

As one of the most important parameters of the MR contrast agent, the MR relaxivity r1 was investigated, as shown in Figure 1F,G. A remarkably high r1 of about 76.7 mM–1 s–1 at 3T was recorded for BSA-Gd, which was nearly 16-fold higher than that of the commercial contrast agent Gd-DTPA (4.7 mM–1 S–1) and 7-fold more robust than that of BSA-Gdx under the same condition. The significantly enhanced relaxivity of BSA-Gd may be attributed to the following reasons: 1) the binding of gadolinium and macromolecular albumin increases the molecular weight of the entire contrast agent, thereby slowing down the rotational motion of the complex, and 2) the high hydrophilicity of undenatured albumin allows water molecules around BSA-Gd to easily expose to gadolinium oxide for inner-sphere water relaxation.26−28Figure 1I presents the kinetics of the MR signal in the blood vessels of rats after the contrast agents were intravenously injected. Under the same dose of the Gd concentration, BSA-Gd reached a much stronger signal and a longer blood circulation time with a t1/2 of about 40 min in comparison to Gd-DTPA (t1/2 = 2 min). As is well-known, besides applying a high magnetic field, the prolonged scanning period enhances the spatial resolution of MRA. As shown by the fan-shape phantom (mimicking different size blood vessels), using a scanning time of 13 min at 3 T, a spatial resolution of up to 200 μm could be realized. Taking advantage of the long circulation time and high r1 of BSA-Gd, clear visualization of blood vessels throughout the body of the rat is shown in Figure 1K, which means that ultrahigh-resolution MRA could be realized.

High-Resolution Imaging

As revealed by the phantom experiments, the prolonged scanning time could dramatically enhance the spatial resolution of MRA under a relatively low magnetic field (3T). Rats were scanned locally to investigate the in vivo performance of BSA-Gd for high-resolution imaging at 3T. To mimic the clinical application scenario better, the chest, neck, and head of the rat were scanned simultaneously. Complicated vascular structures were visualized with intravenous injection of BSA-Gd (Figure 2A,B). For instance, plenty of microvessels with a diameter of about 200 μm could be unambiguously presented (Figure 2). The bilateral carotid arteries and their major branches are clearly shown here. More remarkably, the entire lengths of the clinically important vertebrobasilar artery and the anterior cerebral artery were clearly displayed (Figure 2C). The organs in the lower abdomen, particularly the liver and kidneys, where the blood supply is more abundant, were also examined by BSA-Gd-assisted MRA. Visualizing the kidney vasculature with MRA is well known to be very challenging (Figure S12). Commercial contrast agents are almost excreted through the kidneys; therefore, the imaging of small vessels would be dramatically interfered by the rapidly intensifying renal tubular signal. Due to the liver metabolism properties and the excellent relaxivity of BSA-Gd, the main vessels of the rat kidney could be obviously distinguished up to four orders of branches, including segmental vessels, interlobular vessels, and arcuate vessels (Figure S12). Figure S13 shows the high-resolution images of rat liver vessels with the assistance of BSA-Gd. The portal vein, left hepatic vein, middle hepatic vein, right hepatic vein, and corresponding ultrafine branches could be clearly labeled. Figure S16 shows a clear visualization of the site of arterial occlusion in rats after the injection of BSA-Gd, with the distal end of the occlusion being enhanced due to the presence of collateral circulation.

Figure 2.

Figure 2

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MRA images of the head, neck, and chest of the rat under ultrahigh spatial resolution. Three-dimensional images of the rat head, neck, chest, and forelimbs of sagittal (A) and coronal (B) planes with countless vessels were acquired by 3T MR after being injected with 0.1 mmol kg–1 Gd of BSA-Gd. Small vessels with diameters of 200 μm are presented here (inset). Clinically important brain vessels are shown in MR images (C) and a schematic diagram (D).

Tumor Vessel Labeling

Encouraged by the excellent performance of BSA-Gd in in-vivo high-resolution angiography, we explored its performance in labeling of tumor vessels. The tumor vessels labeling procedure by BSA-Gd at 3T MR is illustrated in Figure 3A. The tumor implanted in the leg muscle exhibited a high intensity signal on T2-weighted imaging (Figure 3B, red circle). As depicted in the contrast-enhanced MRA image after BSA-Gd was injected (Figure 3C), the iliac and femoral vessels were observed to branch directly to nourish the tumor, and the vessel diameter of 200 μm could be recognized. In addition to the origin of the vessels, the density and course of the vessels were clearly identified. Tumor vessels were clearly labeled in vivo via BSA-Gd even under 3T MR, being greatly superior to Gd-DTPA (Figure 3G). These findings are highly significant for the research on tumor development, treatment selection, determination of efficacy, and prognosis.9,29 Taking advantage of the labeling kidney vasculature and tumor vasculature, we examined the labeling effect of BSA-Gd on the kidney in situ tumor vasculature. Preoperative clarification of the peritumor vascular distribution is essential for guiding partial nephrectomy. It facilitates a decrease in bleeding and shortens the operative time, thus preserving the residual renal function.30 To the best of our knowledge, there are no reports on the clinically approved MR contrast agent that has achieved such a kind of high-resolution imaging on the kidney. Figure S14 clearly illustrates the distribution of blood vessels around the endogenous in situ tumor of the kidney with the assistance of BSA-Gd.

Figure 3.

Figure 3

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BSA-Gd and Gd-DTPA assisted MRA labeling tumor vessels. (A) Experimental process of MRA labeling leg tumor vessels. The T2WI image of rat hind limbs bearing the tumor before being injected with the contrast agent (B) and the BSA-Gd-enhanced MRA image (C). An image of the tumor-bearing leg (D) and tumor hematoxylin and eosin (HE)-stained section (E). T2WI image of rat hind limbs bearing the tumor before being injected with Gd-DTPA (F) and contrast-enhanced MRA image (G). The image of the tumor bearing leg (H) and tumor HE stained section (I), scale bar: 2 mm.

Imaging of Large Mammals

The potential clinical application of BSA-Gd was assessed by in vivo MRA studies on New Zealand rabbits and beagles. For high-resolution angiography in rabbits, the brain and lower extremities were investigated, as shown in Figure 4. We could trace the abdominal aorta in the pelvis to the bilateral iliac arteries, which travel downward to the lateral branches to nourish the femur and finally extend to the femoral arteries to nourish the lower extremities (Figure 4A,B). For the cerebral vasculature, as in rats, high-resolution imaging revealed all clinically significant vascular structures, including the anterior cerebral artery, middle cerebral artery, posterior cerebral artery, and cerebral arterial rings (Figure S15). The reconstructed MR image could reach a resolution of capillaries as low as 300 μm via the administration of BSA-Gd (0.12 mmol kg–1) (Figure 4). Nevertheless, Gd-DTPA failed to reveal any significant vascularity under the same Gd concentration, indicating that the performance of BSA-Gd was greatly superior to that of Gd-DTPA (Figures S17 and S18).

Figure 4.

Figure 4

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MRA images of rabbit hind limbs with an ultrahigh spatial resolution. A three-dimensional image of rat hind limbs from the coronal (A) and sagittal plane (B) was acquired by 3T MRA after being injected with 0.12 mmol kg–1 Gd of BSA-Gd. Small vessels with diameters of 300 μm are presented here (inset).

Adult beagles could be considered a kind of large mammal for their weight comparable to that of young children and a high similarity in blood vessel distribution to humans.31 The pelvis and hind limbs of the beagle were scanned locally via intravenous administration of BSA-Gd (Figure 5A). The first temporal image shows the arterial phase, followed by the venous phase, showing progressive signal enhancement over time. A clear distinction was noted between the arteries and the veins (Figure 5B). The whole-body vascular system was recorded for the beagle in a single dose and scan. Surprisingly, the spinal arteries, which are very difficult to observed in clinical MRA, could be clearly marked (Figure 5C).

Figure 5.

Figure 5

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BSA-Gd-enhanced MRA of pelvic cavity regions and whole-body images of the beagle. (A) Dynamic contrast enhancement of MRA images of the beagle pelvic cavity after 0.06 mmol Gd of BSA-Gd was injected. Arteries and veins could be clearly distinguished (inset), and the plot of the vessel signal intensity versus time is shown in (B) (n = 3). (C) Whole-body scan showing the labeling of clinically important vessels such as the anterior spinal artery (black arrow) and posterior intercostal artery (red arrow).

Low Dose Imaging

As discussed above, BSA-Gd as a kind of high r1 contrast agent, was prepared successively, for which the r1 was about 16-fold larger than that of the commercial agent Gd-DTPA. The results revealed that the Gd concentration of BSA-Gd was much lower than that of the Gd-DTPA to achieve the same relaxation rate. Therefore, a low administration dose of BSA-Gd was applied for MRA investigation, for which the dosage was about 1/16 of the recommended clinical dosage of Gd-DTPA. For the BSA-Gd rat model, systemic large vessels were clearly marked including the carotid artery, aortic arch, jugular vein, upper and lower vena cava, subclavian vein, and their branches. However, the outlines of corresponding vessels were dim in rats with an injection of the same Gd concentration of Gd-DTPA (Figure 6B). Moreover, as the r1 of BSA-Gd is nearly 16-fold higher than that of Gd-DTPA, a high dose of Gd-DTPA of about 16-fold higher than that of BSA-Gd was administered as shown in Figure 7C. Under such conditions, the relatively large vessels such as the jugular and inferior vena cava could be visualized, whereas the background signal was also elevated. The rapid and nonspecific tissue diffusion properties of Gd-DTPA made it difficult to recognize small vessels. More importantly, as shown in Figure 7B,C, the heart could not be imaged after 2 min of intravenous administration of Gd-DTPA and suffered from the rapid metabolization of the injected contrast agent. On the other hand, the heart, neck vessels, and brain vessels were still clearly visible 15 min after BSA-Gd injection (Figure 7A). Figure 7D–F demonstrates that BSA-Gd was metabolized by the liver. Therefore, we expected a much longer effective imaging window and found that the vascular-to-muscle contrast for the injection of BSA-Gd was about 3 times higher than that of Gd-DTPA.

Figure 6.

Figure 6

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Comparison of whole-body angiography in rats at low doses of contrast agents. MRA images were scanned after BSA-Gd (A) or Gd-DTPA (B) was injected intravenously at the Gd concentration of 0.05 mmol kg–1. Both groups of rats were scanned for the same parameters. The heart, carotid artery, aorta, aortic arch, jugular vein, subclavian vein, inferior vena cava, and hepatic vein, as well as their branches, were clearly observed in BSA-Gd-injected rats and were very vague in Gd-DTPA-injected rats.

Figure 7.

Figure 7

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Whole-body scan at different time points of rats injected with low doses of BSA-Gd and Gd-DTPA and high doses of Gd-DTPA. Under a 3T MR treatment, rats were injected with BSA-Gd (A; Gd = 0.05 mmol kg–1), Gd-DTPA (B; Gd = 0.05 mmol kg–1), and Gd-DTPA (C; Gd = 0.75 mmol kg–1). Plot of the signal intensity contrast of the vessels (D), liver (E), and bladder (F) with muscles versus time of different doses of agents, n = 1, scale bar = 20 mm.

Metabolic and Biosafety Assessment

Besides exploring the efficacy, the biosafety of nanomaterials should also be taken fully into account. We investigated the toxicity of BSA-Gd for four kinds of noncancerous cell lines in vitro. The cell activity assay indicated that even at the Gd concentration of about 10 mM, the cell viability remained more than 90% after continuous incubation with BSA-Gd for 24 or 48 h, as shown in Figure S11. The Gd3+ leakage experiment detected no free Gd3+ during dialysis for a week, indicating the stability of BSA-Gd in physiological environments (Figure S6). We subsequently obtained rat serum at 10 min after administration and performed TEM, and no significant changes were observed in the size and morphology (Figure S5). Therefore, under our experimental dosage, the cytotoxicity of BSA-Gd was negligible. For biosafety assessment, the pharmacokinetic parameters were considered as one of the key indicators. Therefore, we studied the biological distribution and excretion of intravenous administration of BSA-Gd in rats. Figure S8B reveals that almost all Gd was metabolized through the liver and excreted by the digestive tract; this finding was completely consistent with the enhancement of the MRA signal in the liver and the low signal intensity in the urinary system (Figure S7). On day 30 of scanning, the signal intensity of each organ was consistent with what it had been before administration. Moreover, the absence of Gd in both urine and feces after 30 days suggested that most of the BSA-Gd had been excreted from the body. Further, inductively coupled plasma mass spectrometry (ICP–MS) results showed that the Gd content in organs and the intensity of MR signals were consistent with that after administration, and after 30 days, no Gd was detected in the main organs (Figure S8A). The biocompatibility of BSA-Gd was investigated in vivo by carrying out acute and chronic toxicity assays on six rats per group. Rats were injected with BSA-Gd with a dose of 0.2 mmol Gd/kg of body weight and monitored every day. There was no significant difference in the growth trend of body weight between the experimental group and the control group (Figure S8C), and there was no obvious behavioral abnormality. The blood tests also showed that the function indexes of the liver and kidneys of rats in each group were within the normal range (Figure S10). The histopathological sections 2 weeks after administration are shown in Figure S9. No edema, necrosis, hyperemia, and other pathological changes were observed in all the important organs of the body.

Conclusions

In this study, we has successfully developed BSA-Gd nanoparticles as a contrast agent for MR angiography that boasts exceptional capabilities. Compared to the widely used commercial agent, the BSA-Gd exhibited a dramatically improved performance and showed high-resolution imaging of vessels in rats, rabbits, and beagles. In this study, vessels of sub-200 μm in the normal tissue and tumor could be clearly visualized via 3T MR and thus provide us a powerful tool for studying physiology and disease. Attributed to the natural drug delivery properties of BSA, BSA-Gd exhibits broad prospects for further development as a new platform for theranostics.

Acknowledgments

The authors acknowledge funding from the National Natural Science Foundation of China (no. 81927807) for X.P.Z and Q.C.S., from the National Natural Science Foundation of China (no. 82002707) for Q.C., and from the Natural Science Foundation of Hubei Province of China (no. 2021CFB442) for X.M.L. The authors would like to thank Zhengrong Yin for providing the Biorender account and Jiao Xie for kindly giving cell lines. We also thank Dingxi Liu for his assistance in optimizing scanning sequences.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsami.3c16391.

  • Additional characterization data and experimental details including TEM, XPS graphs, stability tests, biosafety assessments in vitro and in vivo, and MR images (PDF)

Author Contributions

C.C.L., X.M.L., and Z.H.W. designed and performed the experiments. C.C.L., X.M.L., and Q.C.S. wrote the manuscript. Z.C., Y.W.B., and P.Z. participated in part of the animal experiments. H.G.L., C.J.T., and Z.Q.L. provided some useful advice. P.S. provided assistance in sequence optimization. X.P.Z., Q.C.S., X.M.L., and Q.C. conceived the work and obtained funding for this project. All authors discussed and commented on the manuscript. C.L., X.L., and Z.W. have contributed equally to this work.

Author Contributions

Data are available on request from the corresponding author.

The authors declare no competing financial interest.

Supplementary Material

am3c16391_si_001.pdf (3.3MB, pdf)

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