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. 2023 Jun 10;23(12):5497–5505. doi: 10.1021/acs.nanolett.3c00665

Novel Gadolinium-Free Ultrasmall Nanostructured Positive Contrast for Magnetic Resonance Angiography and Imaging

Rodrigo Ken Kawassaki †, Mariana Romano †, Mayara Klimuk Uchiyama †,‡, Roberta Mansini Cardoso †, Maurício S Baptista §, Sandra H P Farsky ∥, Khallil Taverna Chaim ‡,⊥, Robson Raphael Guimarães †, Koiti Araki †,*
PMCID: PMC10312191  PMID: 37300521

Abstract

graphic file with name nl3c00665_0005.jpg

Nanostructured contrast agents are promising alternatives to Gd3+-based chelates in magnetic resonance (MR) imaging techniques. A novel ultrasmall paramagnetic nanoparticle (UPN) was strategically designed to maximize the number of exposed paramagnetic sites and r1 while minimizing r2, by decorating 3 nm titanium dioxide nanoparticles with suitable amounts of iron oxide. Its relaxometric parameters are comparable to those of gadoteric acid (GA) in agar phantoms, and the r2/r1 ratio of 1.38 at 3 T is close to the ideal unitary value. The strong and prolonged contrast enhancement of UPN before renal excretion was confirmed by T1-weighted MR images of Wistar rats after intravenous bolus injection. Those results associated with good biocompatibility indicate its high potential as an alternative blood-pool contrast agent to the GA gold standard for MR angiography, especially for patients with severe renal impairment.

Keywords: ultrasmall nanoparticle, paramagnetic, contrast agent, MRI, iron oxide

Magnetic resonance imaging (MRI) is a noninvasive diagnosis technique that provides detailed anatomical information on soft tissues whose image resolution to detect injuries is further improved by contrast agents (CAs). Their effect is based on the shortening of the longitudinal (spin–lattice, T1) and transverse (spin–spin, T2) relaxation times and can be classified as T1-/positive or T2-/negative CAs depending on the contrast efficiency measured by their longitudinal (r1) and transverse (r2) relaxivities. Positive CAs are those possessing a r2/r1 ratio closer to 1, the ideal value, whereas negative CAs typically exhibit ratios larger than 10.1,2

Among current FDA-approved CAs, the Gd3+-based contrast agents (GBCAs) are the most applied in clinical practice. However, recent studies demonstrated toxicological effects and metal deposition in different organs, raising concerns about the repeated usage of these compounds.3−6 Nanomaterials have been studied as promising alternatives since their properties can be modulated by controlling their physicochemical characteristics.7,8 Among these, the most explored as potential nanocontrast agents are superparamagnetic iron oxide nanoparticles (SPIONs), due to their high biodegradability and nontoxicity in biological systems when compared to GBCAs.9−12 However, despite their much larger intrinsic magnetic moments those materials still are behind conventional GBCAs, as they essentially act as negative CAs (r2/r1 ratios >10).1,8

The recent development of CAs based on ultrasmall IONPs (USIONs <5 nm) brought about a significant improvement since they can behave as T1-CAs.13−17 Previous works have shown that their r1 and r2/r1 ratio are strongly dependent on the nanoparticle size, shape and core crystallinity, as well as surface interactions and aggregation state in the biological environment.1,2,18,19 For instance, in a systematic study, Ferretti et al. demonstrated that 3 nm large zwitterionic-coated USIONs can act as T1-CAs and be excreted by the urinary system, a characteristic analogous to GBCAs.20 Similarly, Groult et al. found that 3.9–4.3 nm large heparin oligosaccharide coated USIONs presented the optimal correlation between r1 and r2/r1. Furthermore, by varying the oligossacharide length and degree of polymerization, it was possible to control the NP biodistribution and blood circulation time in vivo.21 Interestingly, a polyglucose sorbitol carboxymethyl ether coated USION-based formulation registered for treatment of anemia called Ferumoxytol is being explored as off-label alternative to GBCAs in MRI techniques, including MR angiography.22−24 Furthermore, citrate-coated USIONs have been used in preclinical and clinical studies as potential T1-CA substitutes for GBCAs in MR angiography.25−27

The translation to clinical use is still a challenge as other issues also need to be overcome, such as scaling up the preparation process to improve the production cost, colloidal stability issues, and a suitable application protocol and pharmacological parameters. Hence, many efforts are underway in the quest for safer CAs, but no alternative product to GBCAs with comparable clinical performance and similar clinical practice protocol has been consolidated in the market yet. Accordingly, herein is presented a novel class of ultrasmall paramagnetic nanoparticles (UPNs) with adjustable magnetic and MRI-contrast properties by decorating biocompatible ultrasmall titanium dioxide nanoparticles (usTiO2NPs) with suitable amounts of iron oxide. Titanium dioxide has low toxicity and is diamagnetic, and iron(III) oxide has high adherence onto its surface, allowing the preparation of stable ultrasmall paramagnetic nanoparticles suitable as an MRI contrast agent. The properties of such novel engineered nanomaterials were carefully adjusted to overcome the key issues faced by GBCAs and SPION-based products, including no toxic elements and no significant toxicity, thus avoiding nephrotoxicity and metal accumulation. The safety issues were further assessed by carrying out careful systematic stability studies in biological medium and toxicological assays based on cell viability tests. In addition, r2/r1 values were adjusted to be lower than 2 to overcome the limitation of SPIONs as typical T2-CAs, thus enabling T1-contrast images with quality as high as that using GBCAs as demonstrated in vivo. In addition, UPNs are fully compatible with the currently used clinical imaging protocols while providing extended vascular contrast for a period of up to 20 min, which can be convenient in most cases.

The NPs were produced by a nonhydrolytic method and stabilized with selected functionalizing ligands. The size and morphology were determined using dynamic light scattering (DLS) and transmission electron microscopy (TEM) images. TEM images revealed monodisperse and nonagglomerated spherical UPNs with an average core size of ∼3 nm (dark spots, Figure 1a and Figure S1). Its high dispersibility in water was confirmed by DLS. which provided an average size of 3.8 nm (Figure 1b) with no additional peak in the volume-weighted histograms (PdI < 0.3), as expected for monodisperse NPs. A slightly larger diameter by DLS is expected since it measures the hydrodynamic diameter, which includes the functionalizing molecular layer and the hydration layer in addition to the core (2.8 nm by TEM, Figure S1). Finally, given the very small size and high concentration of surface defects, the NPs do not exhibit a crystalline structure, as demonstrated by X-ray diffractometry (XRD) showing only a broad halo characteristic of amorphous materials at around 20° 2θ (Figure 1c). This can be better evaluated by comparison with the XRD pattern of SPIONs, which typically exhibits characteristic diffraction peaks.28,29 Furthermore, elemental analysis by energy dispersive X-ray fluorescence showed the presence of Ti and Fe, indicating that iron oxide might be deposited on the surface of the usTiO2NPs (Figure S2).

Figure 1.

Figure 1

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Characterization and in vitro toxicity studies of UPNs. (a) TEM image showing well-dispersed UPN-Cit nanoparticles, (b) hydrodynamic size distribution histogram by volume-weighted DLS, and (c) X-ray diffractogram of UPN-Cit showing the amorphous structure. (d) UPN colloidal stability in RPMI-1640 cell culture medium supplemented with FBS 10% (v/v) as evaluated by DLS (volume-weighted size distribution) after 0 min, 24 h, and 48 h of incubation. MTT and resazurin cytotoxicity assay of UPNs (0.25 mM Fe) functionalized with citrate (Cit), glycerol-3-phosphate (Gly), o-phosphorylethanolamine (PEA), and tiron (Tir) against (e) HUVEC and (f) RAW 264.7 cell lines after 24 h of incubation. The cell culture medium was used as a negative control (Control) in both assays. ANOVA: *p < 0.05; ***p < 0.0005; ****p < 0.0001.

Moreover, nanomaterials intended for biomedical applications need to be colloidally stable in biological systems, but NPs tend to agglomerate/aggregate as the ionic strength increases or when in the presence of other substances.30 Accordingly, their colloidal stability in biological media, such as cell culture media, needs to be evaluated since aggregated nanoparticles often show negative biological responses (inflammation, ROS, loss of functionality)31−34 and also can induce the coupling of their magnetic moments, influencing the r2/r1 ratio value.19,35,36 Hence, the size distribution of the UPNs functionalized with different ligands (citrate (Cit), glycerol-3-phosphate (Gly), o-phosphorylethanolamine (PEA) or tiron (Tir)) (0.25 mM Fe) in RPMI-1640 cell culture medium supplemented with 10% (v/v) fetal bovine serum (FBS) was monitored by DLS. The volume-weighted histogram showed no change after 24 and 48 h of incubation (Figure 1d), indicating no aggregation.

When administered by intravenous (i.v.) injection, NPs will interact with all blood components (biomolecules, electrolytes, circulating blood cells) and vessel walls (endothelial cells). Their permanence in circulation will depend on those interactions and the clearance systems of the body, such as the mononuclear phagocytic system. The blood clearance can be performed by specialized cells such as tissue macrophages, especially if their size is >5.5 nm.20,37,38 Hence, the in vitro toxicity of UPNs functionalized with the same ligands series was assessed by the MTT and resazurin methods using HUVEC and RAW 264.7 cell lines as relevant models of endothelial cells and macrophages, respectively, by comparing the cell viability after 24 h of exposure to UPNs (0.25 mM Fe) with controls (culture medium). There was no significative reduction on HUVEC cell viability treated with UPNs as compared with controls (p < 0.05) (Figure 1e). In contrast, the treatment with UPNs caused a small reduction on the RAW 264.7 cell viability (Figure 1f) in comparison with control in both MTT and resazurin assays (p > 0.05), but the cell viability remained above 70% in both cases and was not considered potentially cytotoxic according to ISO standards.39

The volume- and intensity-weighted size distribution histograms were compared to make clear the DLS pattern associated with UPNs (Figure 1d and Figure S3a, respectively) and proteins present in the culture medium supplemented with FBS (Figure S3c).40,41 Since UPNs and proteins have similar sizes (3 and 10 nm, respectively), it is reasonable to evaluate the size distribution by volume and by intensity rather than by number to assess the colloidal stability and the eventual formation of a biomolecular corona. Interestingly, the proteins seem to be found in two states according to the intensity-weighted distribution: a dissociated one (∼10 nm) and a much larger one which can be attributed to an associated state involving UPNs and biomolecules (∼100 nm) (Figure S3a). The shift of the peak from about 10 nm (time zero) to 100 nm after 24 and 48 h with a contrasting size distribution pattern relative to control (Figure S3a) clearly indicates the UPN interaction with protein particles favoring the associated state, which can be related to a corona layer but not to aggregation.40,42 Such a process is reproducible and seems to be controlled by specific NP/protein interactions, given their similar negative zeta potentials (ZPs) (Figure S3d). The similar size distribution patterns (Figure S3a) with absence of further macroscopic precipitation (Figure S3b) indicate they are suitable for biological application.

The UPNs functionalized with different ligands presented negative ZPs, with the citrate derivative presenting the lowest ZP. Carboxy dextran and polyethylene glycol are commonly used NP-functionalizing molecules13 and can enhance the blood circulation time and avoid opsonization.10,43−45 However, our previous works indicated that small molecules such as those used in this work are interesting to adjust the colloidal stability and the cellular uptake,46,47 while facilitating the interaction of water molecules to the paramagnetic Fe(III) sites on the UPN surface and thus enhancing T1 relaxation and decreasing the r2/r1 ratio. Moreover, previous studies in vivo and in humans have been reported using citrate-coated USIONs.25,26 Thus, a formulation with 25 mM Fe and 150 mM propylene glycerol, to adjust the osmolarity to 1600 mOsm/kg H2O, was prepared, filtered through a 0.22 μm filter, and sealed in 2 mL sterile amber glass ampules, under good practice conditions, for use in the biological assays. The formulated UPN-Cit had a ZP of −34.7 ± 5.5 mV and formed a nanofluid containing individually dispersed nanoparticles.

The transversal spin relaxivity and magnetization are two related key parameters that are sensitive to the size and degree of crystallinity of SPIONs, which are typical T2-CAs since their much higher magnetization is very effective in generating more extended magnetic inhomogeneities responsible for spin–spin relaxation processes (high r2 and r2/r1 values). In contrast, the longitudinal spin relaxivity responsible for the T1-contrast, is strongly dependent on the direct interaction of the water molecules with the CA paramagnetic sites.1 Thus, GBCAs always have a labile coordination site for interaction with water molecules to optimize r1.4 This implies that smaller nanoparticles with larger surface areas tend to be better suited as alternatives, since increasingly larger amounts of the paramagnetic ion will be exposed at the surface, promoting the spin–lattice relaxation.1 Additionally, paramagnetic ions in nanoparticle surfaces induce as low as possible disturbance on the local magnetic field (lower r2) due to surface spin-canting effects.1,2 This reduces their saturation magnetization since the magnetically dead surface volume relative to its inner core tends to increase as the size decreases.14 Hence, the sum of effects leads to a higher r1, while r2 decreases and the r2/r1 ratio tends to 1. Shen et al. found that 3.6 nm diameter poly(acrylic acid)-stabilized IONPs presented a low r2/r1 ratio (2.6 at 1.5 T and 10.5 at 7 T),15 whereas Kim et al. showed that 1.5 nm large IONPs exhibit a magnetization behavior similar to that of paramagnetic materials.14 The UPNs described herein are usTiO2NPs decorated with paramagnetic iron oxide, which fulfills the requirements for application as MRI CAs.

Gadoteric acid (GA) is the gold standard T1-CA in clinical MRI diagnostics; hence, its MR relaxometric properties were compared with those of the novel UPNs in agar phantoms. Its r1 and r2 values were determined to be 1.76 and 2.43 mM–1 s–1 at 3 T and 1.32 and 2.35 mM–1 s–1 at 7 T, respectively, as compared to 7.32 and 4.84 mM–1 s–1 at 3 T and 3.80 and 3.53 mM–1 s–1 at 7 T of GA, respectively (Figure 2a,b). Thus, the r2/r1 ratios of UPN are 1.38 (3 T) and 1.78 (7 T), in comparison with 0.66 (3 T) and 0.93 (7 T) for GA, typical parameters of T1-CAs. Interestingly, the relaxivity of UPN is much less sensitive to the magnetic field than GA, being promising as CAs in high-field equipment.

Figure 2.

Figure 2

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MR relaxometry of UPN and GA. Comparison of (a) r1 and (b) r2 curves obtained at 3 and 7 T magnetic fields in agar phantoms with increasing concentrations of the paramagnetic ions. (c) T2 and (d) inversion recovery curves at 5 mM paramagnetic ion in a 7 T magnetic field showing the shortening of T2 and T1, respectively. (e) Spin echo images for UPN and GA in agar as compared to agar control acquired at different TR times, emphasizing the hypersignal at low TRs (dashed line red square). CPI: concentration of paramagnetic ion (mM).

Furthermore, the T2 and inversion recovery curves determined for 5 mM of paramagnetic ions in agar at 7 T indicate that UPNs shorten T2 (Figure 2c) and T1 as well (Figure 2d) but less effectively than GA. This is expected considering that Gd3+ (7e–) has a larger number of unpaired electrons than Fe3+ (5e–), but the difference persists even after normalization using such a parameter. The effectiveness of T1 shortening can be seen in Figure 2e, where low TR times result in a hypersignal in the spin echo sequence when compared with the control, showing that the new UPNs can also be classified as T1-CAs.

The UPNs presented much lower r2/r1 ratios at 3 and 7 T (1.38 and 1.78, respectively) than IONPs reported in the literature (Table 1), clearly evidencing the success of our strategy and nanostructure design based on iron oxide decorated usTiO2NPs. Ferumoxytol has an r2/r1 ratio 4.5 times larger than that of UPNs, while Feridex exhibits an even larger value (r2/r1 = 22.7). In short, the controlled deposition of iron oxide on usTiO2NPs can generate CAs with T1 and T2 relaxation characteristics similar to those of GBCAs. The presence of iron oxide only at the surface of usTiO2NPs maximizes the number of paramagnetic sites that can interact directly with water molecules in the medium, promoting spin relaxation by the spin–lattice mechanism while minimizing the spin–spin relaxation.

Table 1. Relaxivities of Commercial MRI Contrast Media Based on Iron Oxide Nanoparticles in Comparison with UPN and Gadoteric Acid (mM–1 s–1).

contrast agent r1 r2 r2/r1 magnetic field (T) ref
Supravist (SHU 555C) 7.3 57 7.8 3 (48)
Feridex/Endorem 4.1 93 22.7 3 (48)
Resovist (SHU 555A) 4.6 143 31.1 3 (48)
Ferumoxytol 10a 62.3a 6.2 3 (49)
Sinerem 6.58b 127.8b 19.4 3 (50)
UPN 1.76 2.43 1.38 3 this work
gadoteric acid (GA) 7.32 4.84 0.66 3 this work
UPN 1.32 2.35 1.78 7 this work
gadoteric acid (GA) 3.80 3.53 0.93 7 this work
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a

Measured in saline solution.

b

Measured in Ficoll solution.

After careful evaluation of the colloidal stability of UPNs in biological media, in vitro cytotoxicity, and efficacy, the potential of the novel UPNs as T1-CAs was further evaluated in vivo using Wistar rats (4 males, 2 for GA group and 2 for UPN group) as animal models, using a 7 T MR scanner. The images were acquired before and after a 20 s i.v. bolus injection of GA (0.1 mmol/kg of body weight, 25 mM of GA) or UPN (0.1 mmol/kg of body weight, 25 mM Fe) following a protocol approved by the FMUSP Animal Ethics Committee (#966/2018). The time-dependent T1-weighted MR images (Figure 3 and Movies S1 and S2) obtained postinjection of the CAs show the enhancement of the brightness in the heart, liver, blood vessels, and kidneys (yellow arrows), in comparison to the images obtained before the injection, as expected. In addition, a dynamic study was also performed. Sequences of MR images were obtained along 5 min after i.v. injection through the penile vein, with a temporal resolution of 1.6 s. The average signal for 2 animals per group was plotted as a function of time, generating time–signal intensity curves (TIC), whose regions of interest (ROI) were manually segmented as shown in the insets. The baseline was corrected considering the signal before the injection of the CAs. The raw signal vs time plots (Figure 4) suggest the similarity of GA and UPN responses in enhancing the T1-signal intensity in heart, kidneys, and liver soon after the injection of the CAs, in agreement with the MR images obtained at 3 min shown in Figure 3. In the case of heart and liver, the signal increases until peaking and then decreases, as expected for the dilution induced by the heartbeat and high vascularization, indicating that both CAs present similar TIC pattens in heart and liver (Figure 4a,c).

Figure 3.

Figure 3

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T1-weighted MR images of Wistar rats before (precontrast) and 3, 11, 16, and 21 min after i.v. bolus injection of (top line) UPN and (bottom line) GA contrast agent. Yellow arrows indicate regions of contrast enhancement after injection of contrast agents: heart, liver, kidneys, and blood vessels. Red arrows show the arrival and accumulation of the contrast agent on the renal calyx at different times. The blue arrow indicates the region of the bladder. Ref.: phantom reference prepared by dissolving UPNs in agar.

Figure 4.

Figure 4

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Time–signal intensity curves. Average of T1-signal intensity of 2 rats as a function of time in the (a) heart, (b) kidneys, and (c) liver, as indicated by the respective ROIs, after a 20 s i.v. bolus injection of GA or UPN.

In the TIC of kidneys, there is a sharp signal increase followed by a late and slower intensity enhancement, probably reflecting the filtration process by nephrons (Figure 4b). A more detailed analysis of the time series of T1-weighted MR images obtained before and after bolus injection of GA and UPN shown in Figure 3 demonstrates the difference in the kidney filtration process of the CAs because of their molecular and nanoparticulate nature, respectively. The progressive accumulation of GA in the renal pelvis after 11 min of injection (red arrow) is expected due to its preferential elimination by the urinary excretion pathway.51 At longer times (16 and 21 min), it is possible to visualize a bright signal at the ureters and finally in the bladder (blue arrow in Figure 3, bottom lane). Accordingly, the UPN also showed a tendency of renal accumulation and excretion but took a much longer time (21 min) after the injection to start appearing in the kidneys (red arrow in Figure 3 top lane). MR images obtained at longer times suggest that the brightness of blood vessels progressively decreases, while the brightness of the bladder was enhanced (Figure S4). The glomerular filtration barrier has a cutoff size of 5–6 nm; thus, smaller particles are expected to be filtered from the blood into the kidneys52 and then eliminated by urinary excretion. Therefore, its late accumulation in the renal pelvis may be attributed to the negative surface charge (Figure S3d) and possible electrostatic repulsion when reaching the glomerular filtration membrane (GFM).52 Another possibility is due to the biomolecular corona effect, which can increase the hydrodynamic size of UPNs (Figure S3a), consequently slowing down the crossing rate through the GFM.52,53 On the other hand, this result also suggests that UPN may have a longer circulation time than GA (Figure 3). This is interesting, since it could be used as a blood-pool contrast agent for MR angiography, a technique in which the vasculature structures are imaged.26,27,54 In fact it is possible to clearly see the blood vessels of the rat even after more than 10 min of acquisition of MR images, as shown in Figure 3. A more biocompatible material55 could reduce the nephrotoxicity in patients with renal deficiency while avoiding systemic nephrogenic fibrosis, since titanium and iron do not undergo transmetalation in vivo in contrast with Gd3+.56

Summarizing, the novel UPN designed as a T1-weighted MR contrast agent is fully compatible with the conventional clinical administration protocol and presented relaxometric parameters comparable to those of GA in agar phantoms as well as a similar image quality after i.v. bolus injection in Wistar rats. The renal elimination rate of UPN was about half that of GA, assuring an exceptional contrast effect for a twice as long time, which can be quite advantageous as a blood-pool contrast agent for MR angiography. Furthermore, the UPN exhibited low cytotoxicity against vascular endothelial cells and macrophages used as model cell lines and a good colloidal stability in biological medium. Those combined features indicate that UPN has great potential as an alternative to GBCAs in MR imaging and angiography, especially for patients with severe renal impairment, using a similar clinical application protocol.

Acknowledgments

The authors acknowledge the financial support granted by Sao Paulo State Research Foundation (FAPESP, grants #2019/02151-2 to R.K.K., #2018/21489-1 to K.A., and #2009/54323-0 to PISA), National Council for Scientific and Technological Development (CNPq, K.A. grants 442599/2019-6, 401581/2016-0, 303137/2016-9, and 402281/2013-6), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES program 33002010191P0), and SisNANO-USP. Special acknowledgement is made to Dr. Helton Nogueira Pereira and Dr. Jefferson Bettini of the National Laboratory of Nanotechnology (LNNano/CNPEM) for the TEM image.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.nanolett.3c00665.

  • Brief description of the experimental methods, UPN size distribution histogram obtained from TEM images, elemental analysis of UPNs by EDX, colloidal stability of UPNs functionalized with different ligands in biological medium, and complementary T1-weighted MR images of rats before and after administration of GA or UPN (PDF)

  • Sequence of T1-weighted MR image series showing a i.v. gadoteric acid bolus injection in a Wistar rat, where it is possible to visualize the brightness enhancement in the blood vessels, heart, liver, and kidneys (MOV)

  • Sequence of T1-weighted MR image series showing a i.v. UPN bolus injection in a Wistar rat, where it is possible to visualize the brightness enhancement in the blood vessels, heart, liver, and kidneys (MOV)

The authors declare no competing financial interest.

Supplementary Material

nl3c00665_si_001.pdf (498KB, pdf)
nl3c00665_si_002.mov (3.1MB, mov)
nl3c00665_si_003.mov (1MB, mov)

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