Effect of Polyol Chain Length on Proton Relaxivity of Gadolinium

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Effect of Polyol Chain Length on Proton Relaxivity of Gadolinium Oxide Nanoparticles for Enhanced Magnetic Resonance Imaging Contrast Anupam Guleria,*,‡ Pranjali Pranjali,‡,† Mukesh Kumar Meher,§,† Anamika Chaturvedi,‡,† Sreemoyee Chakraborti,‡ Ritu Raj,‡ Krishna Mohan Poluri,§,∥ and Dinesh Kumar‡ ‡

Centre of Biomedical Research, SGPGIMS Campus, Lucknow 226014, India Department of Biotechnology and ∥Centre for Nanotechnology, Indian Institute of Technology Roorkee, Roorkee 247667, India

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S Supporting Information *

ABSTRACT: We present the impact of surface modifications on the magnetic resonance imaging (MRI) contrast enhancement abilities of gadolinium oxide nanoparticles. A series of gadolinium oxide nanoparticles surface-coated with polyols of different reductive abilities such as diethylene glycol (DEG), triethylene glycol (TEG), tetraethylene glycol (TeEG), and polyethylene glycol (PEG 200) were synthesized. Particle sizes of synthesized Gd2O3 nanoparticles were found to be in correlation with the chain length of glycol. An enhancement in the in vitro and ex vivo relaxivity of Gd2O3 nanoparticles was revealed with the increase in glycol chain length. Among the various nanosystems, PEG-Gd2O3 has the highest in vitro and ex vivo relaxivities and excellent biocompatibility as revealed from cellular cytotoxicity experiments. The enhancement in MR contrast with glycol chain length can be attributed to the increase in surface hydrophilicity, and its modulation can be exploited as a novel strategy for enhancing the MRI contrast of gadolinium-based contrast agents.

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nanoparticles in a diethylene glycol medium,3,5 Ahmad et al. and Kattel et al. synthesized Gd2O3 nanoparticles in triethylene glycol.10,11 However, to the best of our knowledge, no report is available, which investigates the effect of choice of different glycols on the morphology, magnetic, and relaxometric properties of developed nanoparticles, which requires systematic investigation. The present study is envisaged to fill this lacuna, and a series of gadolinium oxide nanoparticles coated with different polyols were synthesized. We have used four types of polyols with different reductive abilities,12,13 that is, diethylene glycol (DEG), triethylene glycol (TEG), tetraethylene glycol (TeEG), and polyethylene glycol (PEG 200). The influence of various polyols on the size and morphology of prepared Gd2O3 nanoparticles is presented. The developed nanoparticles were then systematically investigated for their relaxivities and image contrast enhancement properties by performing in vitro and ex vivo MRI experiments. The results demonstrated an important role of surface chemistry in proton relaxation mechanisms and revealed the improvement of relaxivity and hence the image contrast with proper surface modification.

INTRODUCTION Magnetic resonance imaging (MRI) has become one of the most widely used imaging modality for diagnostic imaging. The sensitivity of this imaging modality can further be enhanced through the administration of MRI contrast agents. Recently, magnetic nanoparticle (MNP)-based contrast agents have been investigated intensively in this direction as they offer very attractive possibilities in MR imaging due to their large surface area to volume ratio, increased payloads of paramagnetic metal ions on the surface, unique magnetic properties, and longer lifetime in the body.1 The most evident candidates for nanoparticulate T1 contrast agents are systems with a large number of unpaired electrons such as those containing Gd3+, Mn2+, and Fe3+ ions.2 Among the various MNPs developed so far, gadolinium-based nanoparticles (Gd2O3 MNPs) are being investigated the most due to their excellent paramagnetic properties with a large effective magnetic moment of 7.9 μB, which in turn strongly accelerate the relaxation of protons leading to a higher relaxation rate.3−6 However, sparse reports are available on Gd2O3 nanoparticles, which investigate the effect of surface chemistry on their magnetic and relaxation properties. Recently, many groups have reported the polyol synthesis of Gd2O3 nanoparticles using different polyols.3,6−11 For instance, while Ahrén et al. and Engström et al. prepared the Gd2O3 © XXXX American Chemical Society

Received: May 1, 2019

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DOI: 10.1021/acs.jpcc.9b04089 J. Phys. Chem. C XXXX, XXX, XXX−XXX

Article

The Journal of Physical Chemistry C

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Fourier Transform Infrared (FT-IR) Spectroscopy. To record the FT-IR absorption spectrum, pellets of dried powder samples in KBr were prepared, and spectra were recorded between 450 and 4000 cm−1. Magnetic Measurements. The magnetic properties of the synthesized nanoparticles were measured in a superconducting quantum interference device (SQUID) magnetometer (MPMS, Quantum Design). The zero-field-cooled (ZFC) and field-cooled (FC) temperature dependences of magnetization were carried out in an applied magnetic field of 100 Oe between 2 to 340 K. Magnetization (M) versus applied field (H) (i.e., M−H) curves were also recorded at 5 and 300 K in a magnetic field range of −50 to 50 KOe. Relaxivity Measurements. Both the T1 and T2 relaxation times as well as R1 and R2 map images were measured using a 3 T MRI instrument (Siemens Skyra) equipped with a head coil. To acquire the T1 and T2 weighted in vitro and ex vivo phantom images, dispersions of DEG-, TEG-, TeEG-, and PEG-Gd2O3 nanoparticles were prepared in water and fetal bovine serum (FBS), respectively. The metal concentrations in the dispersions were varied from 0.1 to 1 mM. The T1 and T2 pulse sequence parameters were the same for both in vitro and ex vivo MR imaging. For T1 measurements, an inversion recovery pulse sequence was used with the following parameters: field of view (FOV) = 15 cm, phase FOV = 1, matrix size = 256 × 320, slice thickness = 2 mm, spacing gap = 0, repetition time (TR) = 5000 ms, time to echo (TE) = 12 ms, and the inversion time (TI) was varied from 100 to 4000 ms. Net MRI signal amplitude data for each sample were then determined from the signal intensity of the appropriate region of interest (ROI) at each TI using the Siemens software and fitted to the following nonlinear least-square regression (eq 1) to determine the corresponding relaxation time

EXPERIMENTAL SECTION Materials. Gadolinium(III) chloride hexahydrate (GdCl3· 6H2O, 99.99%) and sodium hydroxide pellets (NaOH, 98%) were procured from Alfa Aesar. Diethylene glycol (99%), triethylene glycol (98%), tetraethylene glycol (99%), and polyethylene glycol purchased from Loba Chemie were used as solvents. All chemicals were of analytical grade and used as received without any further modifications. Deionized water was used during all the synthesis procedures and to prepare the aqueous sample solutions. Fetal bovine serum (South American origin) procured from MP Biomedicals was used for ex vivo MRI studies. For cell culture experiments, Dulbecco’s Modified Eagle’s Medium (DMEM) (Himedia ALL11118X), phosphate buffer saline (PBS) (Himedia TL1101), Pen-Strep (Himedia A001-5X), and fetal bovine serum (FBS) (Himedia RM10409) were purchased from Himedia. For the MTT assay, 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT reagent) (SRL 33611) was purchased from Sisco Research Laboratories Pvt. Ltd., and dimethyl sulfoxide (DMSO) (Sigma D8418) was procured from Sigma-Aldrich. Bare gadolinium oxide nanopowder (20− 40 nm, catalog no. 44020) was also procured from Alfa Aesar for comparison. Synthesis of Gd2O3 Nanoparticles. Gd2O3 nanoparticles were synthesized via the polyol route using different polyols such as diethylene (DEG), triethylene (TEG), tetraethylene (TeEG), and polyethylene glycol (PEG 200) (Figure S1). In a typical synthesis, a precursor solution of 5 mmol of gadolinium(III) chloride hexahydrate was prepared in 30 mL of polyols in a round-bottom flask, which was then heated to 80 °C with magnetic stirring until the precursor was completely dissolved in the solvent. Then, a 15 mmol NaOH solution made in 10 mL of polyol was added to the precursor solution. The reaction temperature was slowly increased to 180 °C, and the reaction mixture was kept at this temperature for 4 h. The mixtures were allowed to cool at room temperature and were centrifuged at 2700 rpm for 40 min. The supernatant was decanted, and the nanoparticles settled down at the bottom were washed with double distillation three to four times to remove the excess ions followed by membrane dialysis overnight. The collected nanoparticles were further dried out in air, and the obtained powder was used for further characterizations. Transmission Electron Microscopy (TEM). High-resolution images of Gd2O3 nanoparticles were recorded with a Tecnai G2 20 S-TWIN transmission electron microscope (TEM) (FEI, Netherlands) operated at 200 kV. Samples were prepared by depositing two to three droplets of the dilute solution of the nanoparticles onto carbon-coated copper grids (Icon Analytical Equipment Pvt. Ltd.). Energy-dispersive X-ray spectroscopy (EDX) was also carried out in situ using the embedded detectors of the same instrument. Scanning Electron Microscopy (SEM). A field emission scanning electron microscope (Carl Zeiss FESEM) was also used to study the morphological features of nanoparticles. X-ray Diffraction. Gd2O3 NPs were also characterized using powder X-ray diffraction (XRD) to determine the crystal structure and purity. XRD was carried out with a Bruker D8 Xray diffractometer equipped with a Cu Kα1 source at 40 kV and 30 mA. Gd2O3 NPs were placed in a glass holder and scanned from 10° to 90° with a scanning rate of 2.0°/min.

M TI = M 0(1 − 2e−TI/ T1)

(1)

where MTI is the MRI signal intensity of a selected ROI at inversion time TI. Fitted parameters M0 and T1 are the MRI signal amplitude at TI = 0 and the concentration-dependent relaxation time of the agent, respectively, which were determined simultaneously for each concentration. For T2 measurements, a multi echo fast spin-echo sequence was used simultaneously to collect a series images at different echo times. The parameters used were as follows: matrix size = 410 × 512, repetition time (TR) = 6000 ms, and the spin echo time (TE) was varied from 10 to 230 ms. Other parameters were similar to the ones given for the inversion recovery sequence. The T2 relaxation times were calculated by fitting the obtained magnetization amplitude data at each TE with the following multiparametric nonlinear least-square regression (eq 2) M TE = M 0(e−TE/ T2)

(2)

where MTE is the MRI signal intensity of a selected ROI at echo time TE. M0 and T2 are the MRI signal amplitude at TE = 0 and the concentration-dependent relaxation time of the agent, respectively, which were determined simultaneously for each concentration. The r1 and r2 relaxivities for each sample were then determined from the slopes of the plots of relaxation rates 1/T1 and 1/T2 against the Gd concentration, respectively. The R1 and R2 maps were generated from the corresponding T1 and T2 maps obtained from Siemens syngo MMWP VE36A software. The color mapping of the grayscale MR images was B

DOI: 10.1021/acs.jpcc.9b04089 J. Phys. Chem. C XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry C

Figure 1. X-ray diffraction patterns of Gd2O3 nanoparticles synthesized in (A) DEG, (B) TEG, (C) TeEG, and (D) PEG (“as prepared” and “after calcination” stand for as synthesized (without calcination) and calcined (at 800 °C) nanoparticles).

Figure 2. (A−D) TEM and (E−H) SEM images of as-prepared (uncalcinated) DEG-, TEG-, TeEG-, and PEG-Gd2O3 nanoparticles, respectively. The particle size distribution histograms determined from the SEM micrographs are displayed in (I)−(L). Red solid lines are the Gaussian fitting to the distribution.

Cytotoxicity Assessment. The cellular toxicity of the synthesized nanosystems was assessed by an MTT assay with human embryonic kidney cell lines (HEK 293T ATCC). Since

carried out using a MATLAB (The Mathworks Inc.) program, and the “Jet” color scheme was chosen to represent the MRI results. C

DOI: 10.1021/acs.jpcc.9b04089 J. Phys. Chem. C XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry C Table 1. In Vitro and Ex Vivo r1 and r2 Relaxivities of DEG-, TEG-, TeEG-, and PEG-Gd2O3 Nanoparticles nanosystem DEG-Gd2O3 TEG-Gd2O3 TeEG-Gd2O3 PEG-Gd2O3

particle size (nm)

in vitro r1 (mM−1 s−1)

in vitro r2 (mM−1 s−1)

in vitro r2/r1

ex vivo r1 (mM−1 s−1)

ex vivo r2 (mM−1 s−1)

± ± ± ±

1.14 2.60 3.99 5.75

13.5 16.4 22.6 28.7

11.8 6.31 5.67 4.99

0.09 0.38 0.63 0.99

5.52 8.74 9.86 12.4

13 16 19 21

2 2 3 4

Figure 3. Magnetization (M−H) curves for the DEG-, TEG-, TeEG-, and PEG-Gd2O3 nanoparticles at (A) 5 K and (B) 300 K. (C) and (D) show the temperature dependence of magnetization (M−T) for DEG- and TEG-Gd2O3 and TeEG- and PEG-Gd2O3 nanoparticles, respectively, at H = 100 Oe.

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RESULTS AND DISCUSSION The phase purity and crystallinity of as-synthesized Gd2O3 nanoparticles (NPs) were investigated using X-ray diffraction (XRD) patterns as shown in Figure 1A−D. Each XRD pattern exhibits a broad peak at ∼29°, which corresponds to the (222) crystal planes of cubic Gd2O3, the most intense peak of this oxide. The observed peak broadening and low intensity is most likely due to the small size of the nanocrystals; a similar XRD pattern has been observed by Kattel et al. for ultrasmall Gd2O3 NPs.10,11 All powder samples were then calcinated in a muffle furnace at 800 °C in air for 8 h to further confirm the phase purity. After calcination, explicit XRD diffraction peaks were observed as shown in Figure 1A−D due to improved crystallinity resulting from crystallite growth. Generally, during the heating process, the particles collide and coalesce with one another resulting in larger crystallite sizes.19 It is revealed that the calcined TeEG- and PEG-Gd2O3 nanoparticles exhibit sharper XRD peaks as compared to DEG- and TEG-Gd2O3 nanoparticles. Moreover, the sharpness increases as we move toward the higher chain length polyol suggesting the increase in particle size of calcined samples with glycol chain length. The observed peaks can be well indexed to (222), (400), (440), and (622) crystal planes of the pure cubic Gd2O3 phase corresponding to the standard card (JCPDS no. 43-1014). No additional peaks were seen indicating the formation of a purely cubic Gd2O3 phase.

most of the nanosystems exhibit renal clearance and the kidney being an important excretory organ filters bodily fluids and removes metabolic waste products, thus any exposure of nanoparticles to the kidney may affect its renal function.14,15 The HEK 293 cell lines derived from the human embryonic kidney cells have commonly been used as the model cell line for investigating the cytotoxic effects of nanoparticles.16−18 Therefore, the same has been employed in the present study for evaluating the cytotoxicity of synthesized nanoparticles. The HEK cells were cultured in a DMEM medium containing 10% FBS and Pen-Strep and incubated at 37 °C. Cells were seeded in a 96-well plate at a density of 104 cells per well and allowed to adhere overnight in full medium and then switched to low-serum media followed by exposure to the nanoparticles. Cell loading with nanoparticles was carried out at various concentrations up to 1 mM following incubation at 37 °C for the next 24 h. Following the treatment, each well of the plate was added with 0.5 mg/mL MTT and incubated for an additional 3 h. The cell culture media was then removed, and DMSO was added into each well and mixed thoroughly to dissolve formazan crystals. The absorbance was read at 570 nm for the optical density using a Readwell TOUCH ELISA plate analyzer (ROBONIK INDIA Pvt. Ltd.). The absorbance of cells exposed to the medium only (without any treatment) was taken as 100% cell viability (control). All experiments were repeated four times. D

DOI: 10.1021/acs.jpcc.9b04089 J. Phys. Chem. C XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry C

Figure 4. FT-IR absorption spectra of (A) DEG-, (B) TEG-, (C) TeEG-, and (D) PEG-Gd2O3 nanoparticles along with their respective glycols.

absence of hysteresis in the M−H plot is in accordance with the fact that no magnetic transitions were seen in zero-fieldcooled (ZFC) and field-cooled (FC) temperature dependences of magnetization (M−T plots) as shown in Figure 3C,D. Further, no evident split of ZFC and FC curves was observed down to 2 K, indicating the paramagnetic nature of NPs and which is in agreement with previous studies on Gd2O3 nanocrystals.8,10 The temperature dependence of magnetic susceptibility exhibits Curie−Weiss behavior for all nanosystems as shown in Figure S3. The black solid lines in the plots show the fit to the Curie−Weiss equation, which gives the Curie−Weiss temperature, θP, equal to −3.1, −3.0, −2.6, and −2.7 K for DEG-, TEG-, TeEG-, and PEG-Gd2O3 NPs, respectively. The values of θP are close to zero for all the samples, which further demonstrate the characteristic paramagnetic behavior of NPs. Magnetic measurements do not reveal any significant differences in magnetization of DEG-, TEG-, TeEG-, and PEG-Gd2O3 nanoparticles. Figure 4A−D shows the FT-IR absorption spectra of DEG-, TEG-, TeEG-, and PEG-Gd2O3 NPs, respectively, along with their respective polyol. The FTIR spectra of polyols alone are characterized by a number of characteristic bands occurring at 3390−3380 cm−1 attributed to O−H stretching vibration; peaks at 2930 and 2880 cm−1 correspond to the C−H stretching vibration mode of methyl groups of a polymer; bands at 1456 and 1350 cm−1 are due to CH2 bending and wagging vibrations, and peaks between 1130 and 1050 cm−1 are due to C−O and C−O−C stretching vibrations. The polyol-coated Gd2O3 NPs exhibit a broad band centered at 3425−3410 cm−1 due to O−H stretching vibration arising from the surface hydroxyl groups of adsorbed glycols and water molecules.21 A clear shift in the O−H stretching band (∼30− 35 cm−1) of the polyol-coated Gd2O3 NPs to a higher wavenumber compared to their respective polyols was observed confirming the formation of hydrogen bonds between glycols and Gd2O3 NPs. The characteristic peak observed near 690 cm−1 for all the samples corresponds to the

The morphology and particle size of the nanoparticles were determined using transmission and scanning electron microscopies. Figure 2A−D shows the TEM images of as-prepared DEG-, TEG-, TeEG-, and PEG-Gd 2 O 3 nanoparticles, respectively. TEM analysis revealed that all the Gd2O3 nanosystems consist of globular nanoparticles and their agglomerates with DEG-Gd2O3 having the smallest particle size. Scanning electron micrographs (SEM) of the as-prepared DEG-, TEG-, TeEG-, and PEG-Gd2O3 nanoparticles are shown in Figure 2E−H, respectively. The particle size distributions were determined by image analysis of SEM micrographs by measuring the diameter of approximately 200 particles using the ImageJ software (http://rsbweb.nih.gov/ij/ ). The histograms of particle diameters versus the number of particles are shown in Figure 2I−L for DEG-, TEG-, TeEG-, and PEG-Gd2O3 nanoparticles, respectively. The average diameters of the nanoparticles obtained from the fit of a Gaussian function to the histograms are enlisted in Table 1. We find that particle size increases with the increase in chain length of glycols demonstrating a linear correlation between the length of glycol chain and size of NPs. It seems that polyols having a shorter chain length with bulkier hydroxyl groups are more effective as capping ligands to arrest growth and hence lead to smaller NPs. A similar correlation of NP size with glycol chain length has been reported for iron oxide nanoparticles synthesized in various polyols.20 The TEM images of calcinated DEG-, TEG-, TeEG-, and PEG-Gd2O3 nanoparticles have also been shown in Figure S2 along with their particle size distribution. A clear increase in particle size was seen in all the nanoparticle systems after calcination and exhibits a correlation with the glycol chain length (i.e., the size increases with glycol chain length). Figure 3A,B shows the magnetization of DEG-, TEG-, TeEG-, and PEG-Gd2O3 nanoparticles as a function of the magnetic field acquired at 5 and 300 K, respectively. Both the coercivity and remanence are zero at 5 and 300 K since no hysteresis was observed for all the as-prepared samples. The E

DOI: 10.1021/acs.jpcc.9b04089 J. Phys. Chem. C XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry C stretching vibration of Gd−O indicating the formation of Gd2O3. The coating of polyols on the surfaces of Gd2O3 nanoparticles was further evidenced by the presence of glycolassociated IR bands, such as C−H stretching peaks at 2930 and 2880 cm−1, in-plane C−H bending vibrations at 1400 cm−1, and C−O−C and C−O stretching peaks between 1070 and 1110 cm−1. While the IR spectra of TeEG-Gd2O3 and PEG-Gd2O3 NPs showed intensive bands at around 2930 and 2880 cm−1 attributed to the asymmetric and symmetric C−H stretching modes of −CH2− groups, these bands are very weak for DEG- and TEG-Gd2O3 NPs. The bands located at 1510 and 1400 cm−1 corresponding to CH2 bending and wagging vibrations are also shifted from their original position at 1456 and 1352 cm−1 in pure polyols, respectively. The peaks at 850 and 920 cm−1 can be attributed to C−CH aliphatic deformation vibration and out-of-plane bending vibration of the C−H bond of the CH2 group, respectively. The most important absorption band observed around 1070−1110 cm−1 due to the stretching of C−O−C and C−O bonds in the −CH2−O−CH2− group of glycols for all the four nanosystems is the typical signal of the polyol, which further confirms the successful coatings. It is revealed that the C−O−C/C−O stretching vibration band shifts toward a higher frequency as we go from DEG-Gd2O3 toward PEG-Gd2O3 NPs along with the increase in the intensity of the band. The area under this peak (AUP) has been found to be increased with the increase in the glycol chain length (AUP is 8.68, 9.57, 15.70, and 18.43 for DEG-, TEG-, TeEG-, and PEG-Gd2O3 NPs, respectively) suggesting a correlation between the duo. Therefore, the above-demonstrated results clearly confirm the bonding of different glycols with varied interactive strengths on the surfaces of Gd2O3 nanoparticles. The in vitro proton relaxivity measurements were performed systemically on all the samples to evaluate the effect of surface chemistry of various glycol-coated Gd2O3 NPs on the contrast for MR imaging and hence on r1 and r2 relaxivities. Figure 5A,B shows the plots of longitudinal and transverse MRI signal intensities as functions of inversion time and echo time, respectively, for water and sample solutions of DEG-, TEG-, TeEG-, and PEG-Gd2O3 nanoparticles in water having 0.6 mM Gd concentration. The dotted lines through the data in Figure 5A,B are the fit to eqs 1 and 2, respectively. It is clear from the figures that among all the samples, PEG-Gd2O3 nanoparticles have faster longitudinal magnetization recovery and transverse magnetization decay suggesting higher T1 and T2 relaxation rates. Figure 5C,D shows the color-mapped T1 and T2 weighted in vitro phantom images containing dispersions of DEG-, TEG-, TeEG-, and PEG-Gd2O3 nanoparticles in water, respectively, obtained at the inversion time of 700 ms for T1 weighted images and at an echo time of 107 ms for T2 weighted images. The concentrations of the dispersions were varied from 0.1 to 1.0 mM. A gradual increase in the MR signal amplitude with the increased concentration of metal is seen in T1 weighted images of all the nanosystems (Figure 5C). Similarly, a steady depletion in the MR signal intensity for T2 weighted images was observed with the increase in metal concentration (Figure 5D). Among all the samples, the in vitro phantom images of PEG-Gd2O3 nanoparticles shows the highest signal enhancement and depletion in T1 and T2 weighted images, respectively. The longitudinal and transverse relaxivity values (r1 and r2) for the nanoparticles were determined according to the linear

Figure 5. Plots of (A) longitudinal (T1) and (B) transverse (T2) relaxation signal intensities as functions of inversion time and echo time, respectively, for pure water and sample solutions of DEG-, TEG-, TeEG-, and PEG-Gd2O3 nanoparticles in water having 0.6 mM Gd concentration. Color-mapped (C) T1 and (D) T2 weighted in vitro phantom MR images of DEG-, TEG-, TeEG-, and PEG-Gd2O3 nanoparticles in water with different Gd concentrations.

relationship of longitudinal and transverse relaxation rates versus the metal concentrations as given below: 1 1 = + rC i = 1, 2 i Ti Ti(0) (3) where Ti is the observed relaxation time in the presence of NPs, Ti(0) is the diamagnetic contribution to the relaxation time, ri is the relaxivity constant, which is obtained from the slope of the linear dependence, and C is the Gd concentration. Figure 6A,B depicts the in vitro T1- and T2-based relaxation rates for DEG-, TEG-, TeEG-, and PEG-Gd2O3 nanoparticles as functions of Gd concentration. The corresponding r1 and r2 in vitro relaxivities of DEG-, TEG-, TeEG-, and PEG-Gd2O3 nanoparticles are listed in Table 1. Among the various polyols used, PEG-Gd 2O 3 nanoparticles exhibited the highest relaxation enhancement, with an r1 value of 5.75 mM−1 s−1 and r2 value of 28.7 mM−1 s−1 at 3 T. Both the r1 and r2 values show linear correlation with the glycol chain length and increase with the increase in chain length. The r2/r1 values of all the nanosystems were also calculated and are enlisted in Table 1. It has been reported previously that agents with a moderate r2/r1 ratio (between 3 and 10) can serve as T1−T2 dual-mode contrast agents, while higher (>10) and lower (