Encapsulation of Gadolinium Oxide Nanoparticle (Gd2O3) Contrasting

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Encapsulation of Gadolinium Oxide Nanoparticle (Gd2O3) Contrasting Agents in PAMAM Dendrimer Templates for Enhanced Magnetic Resonance Imaging In Vivo Shewaye Lakew Mekuria, Tilahun Ayane Debele, and Hsieh-Chih Tsai ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b14075 • Publication Date (Web): 06 Feb 2017 Downloaded from http://pubs.acs.org on February 9, 2017

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ACS Applied Materials & Interfaces

Encapsulation of Gadolinium Oxide Nanoparticle (Gd2O3) Contrasting Agents in PAMAM Dendrimer Templates for Enhanced Magnetic Resonance Imaging In Vivo Shewaye Lakew Mekuria, Tilahun Ayane Debele, Hsieh-Chih Tsai* Graduate Institute of Applied Science and Technology, National Taiwan University of Science and Technology, Taipei 106, Taiwan, ROC.

[*] To whom correspondence and reprint requests should be addressed. Prof. Hsieh-Chih Tsai E-mail: [email protected] Tel: +886-2-27303625 Fax: +886-2-27303733

ACS Paragon Plus Environment


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ABSTRACT: There has been growing interest in the research of nanomaterials for bio-medical applications in recent decades. Herein, a simple approach to synthesize the G4.5-Gd2O3poly(ethylene glycol) (G4.5-Gd2O3-PEG) nanoparticles (NPs) that demonstrate potential dual (T1 and T2) contrasting agents in magnetic resonance imaging (MRI) has been reported in this study. Compared to the clinically popular Gd-DTPA contrasting agents, G4.5-Gd2O3-PEG NPs exhibited a longer longitudinal relaxation time (T1) and better biocompatibility when incubated with macrophage cell line RAW264.7 in vitro. Furthermore, the longitudinal relaxivity (r1) of G4.5-Gd2O3-PEG NPs was 53.9 s-1mM-1 at 7T, which is equivalent to 4.8 times greater than to the Gd-DTPA contrasting agents. An in vivo T1-weighted MRI results revealed that G4.5-Gd2O3-PEG NPs significantly enhanced signals in the intestines, kidney, liver, bladder and spleen. However, the T2-weighted MRI results revealed that darker contrast in the kidney, which proves that G4.5Gd2O3-PEG NPs can be exploited as T1 and T2 contrasting agents. In summary, these findings suggest that the G4.5-Gd2O3-PEG NPs synthesized by an alternative approach can be used as dual MRI contrasting agents. KEYWORDS: Gadolinium oxide (Gd2O3), PAMAM dendrimer, longitudinal relaxation, in vivo, T1-T2 MR imaging


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1. INTRODUCTION Magnetic resonance imaging (MRI) is an intensively studied and noninvasive diagnostic technique that applies magnetic field and pulses of radio frequency to produce detailed pictures of organs and internal body structures.1-4 Magnetic nanoparticles have been exploited for biomedical imaging; among these, paramagnetic MRI contrasting agents consisting of gadolinium (III) ions (Gd3+) are able to reduce the longitudinal relaxation time (T1) of the surrounding water protons.5 Furthermore, their performance is enhanced by their seven unpaired electrons, large magnetic moment, and long electronic relaxation time.6-7 However, most commonly used paramagnetic chelating contrast agents, such as Gd (III)-DTPA and their derivatives, are low in molecular weight and exhibit low signal enhancing effects per molecule. Moreover, as a consequence of their small size, many of these agents distribute through intravascular and interstitial spaces, and are rapidly cleared via renal filtration.8-9 In addition, Gd-chelates may release gadolinium ions, that inhibit calcium channels and are associated with nephrogenic system fibrosis

10-11

, that

includes fibrosis of the skin and internal organs in patients with renal insufficiency.12 Currently, high molecular weight macromolecules have been explored as potential platforms for Gd-based agents.13-21 However, the leaching of free Gd from macromolecules remains a critical concern. To resolve this challenge, studies have identified various Gd-based inorganic nanoparticles (NPs) that exhibit higher relaxivity (r1 and r2) and excellent Gd3+ ion retaining ability.22-23 Among these, gadolinium oxide (Gd2O3) nanoparticles (NPs), as well as various gadolinium hybrid nanostructures, have been shown to exhibit superior diagnostic and therapeutic properties.24-29 For example, Gd2O3 incorporated in mesoporous silica nanoparticles (Gd2O3-SBA-15 and Gd2O3-MCM-41 with AuNPs),30-31 bovine serum albumin (Gd2O3-BSA with AuNPs),26 and a polysiloxane shell32 have all been shown to function as efficient contrasting agents in enhanced MRI. In general, the incorporation of gadolinium onto or into nanoscale carriers improves the relaxivity (r1) of MRI agents.8-9, 33 Furthermore, other studies have shown that Gd2O3 exhibits promising T1-weighted effects suitable for T1-positive MRI.34-35 However, single modal (T1 or T2) MRI contrasting agents pose a high risk of false positive signals in the diagnosis of lesions due to the intrinsic background in adjacent tissues. For example, bright signals observed in T1-weighted MRI may be confused with signals in neighboring fat tissues, and the darker signals obtained in T2-weighted MRI may be indistinguishable from background



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signals.36-37 Thus, it is important to synthesize dual-modal (T1/T2) MRI contrasting agents for use in clinical diagnosis. Several researchers have prepared gadolinium-dysprosium oxide and Gd2O3 blended with SPION nanoparticles for use as dual-modal (T1 and T2) MRI contrasting agents.38-41 In this study, we attempted to develop a multifunctional PAMAM dendrimer template for the growth of MRI agents (Gd2O3 NPs) to target normal animal organs in vivo. Dendrimers are versatile polymer with tremendous potential as MRI contrast agents due to their high molecular weight, water solubility, and monodispersity.18, 42-43 In addition, dendrimer-based macromolecular MRI contrasting agents with a range of sizes that provide sufficient contrast enhancement for various applications have been reported.9, 44-46 Furthermore, the high number of functional groups present on the surface of PAMAM dendrimer can be utilized to incorporate paramagnetic materials for MRI contrasting agents.47 For example, poly-(ethylene glycol) (PEG) is commonly introduced into the PAMAM dendrimer structure to improve biocompatibility, prolong half-life, increase circulation time in blood, improve long-term colloidal stability, and enhance structural stability.48-51 Therefore, the dendrimer-based contrasting agents can be used for various MRI applications, including T1 and T2-weighted MRI.52

2. EXPERIMENTAL SECTION 2.1.

Materials: Gadolinium chloride hexahydrate (99.99%) and sodium hydroxide

(99.99%) were purchased from Sigma Aldrich. Ethylene diamine core carboxyl-terminated G4.5 PAMAM dendrimer (G4.5-COOH) was purchased from Dendritech Inc. O-(2-Aminoethyl) polyethylene glycol (Mp 10,000) was from Sigma Aldrich. N-hydroxyl succinimide (NHS) and 1(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) were purchased from Acros Organics (Geel, Belgium). Cellulose dialysis membranes (molecular cutoff of 6000–8000 and 12000–14000 Da) were acquired from CelluSept T1 (Braine-l' Alleud, Belgium). RAW 264.7 cells (mouse monocyte macrophages) were obtained from the Bioresource Collection and Research Center (Hsinchu, Taiwan). Water used in this study was purified using a Milli-Q Plus 185 system and had a resistivity higher than 18.2 mΩ cm. All other chemicals and organic solvents were of HPLC grade. 2.2.

Synthesis of G4.5-Gd2O3 Nanoparticles (NPs): Typically, a 1.5-mM G4.5

PAMAM dendrimer solution was prepared from a stock solution and acidified with 1 N HCl. Then, 4 ml of a 6.5, 13.7, or 27.5 mM GdCl3 solution was added and stirred vigorously to provide


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solutions with final molar ratios of 1:5, 1:10, and 1:20 of G4.5:GdCl3. After 5 min, 2 ml of 3M NaOH was added to each mixture and the mixtures were vigorously stirred at 37°C overnight. The G4.5-Gd2O3 nanoparticles were dialyzed with a cellular membrane (6000–8000 Da) against distilled water for 24 h to remove excess precursors. Final solutions were freeze-dried and concentrated by microfiltration. 2.3.

Preparation of G4.5-PEG Conjugates: For the synthesis of PEGylated

dendrimer conjugates, G4.5 PAMAM dendrimer (10 mg, 0.4 µmol) was dissolved in 2 ml of ultra-distilled water and acidified with 1 N hydrochloride acid. Then, 5 mg of NHS and 9 mg of EDC were added and stirred for 14 h. NHS-activated G4.5 was dialyzed, dried, and re-dissolved in a sodium bicarbonate solution (pH 8.5). Next, O-(2-Aminoethyl) polyethylene glycol (HOPEG-NH2) (0.1 g, 10 µmol) pre-dissolved in a sodium bicarbonate solution (pH 8.5) was added to the G4.5-NHS solution under continuous stirring. The mixture was stirred overnight to initiate the coupling chemistry. The resulting conjugates were extensively subjected to dialysis using a cellular membrane with cut-off of 12–14 kDa against distilled water (1 l), and the outer phase was also replenished with 1 l of new distilled water every two hours. Then, the final solution was lyophilized overnight in a freeze dryer to obtain the final product. 2.4.

Grafting of G4.5-Gd2O3 NPs with PEG: PEG was conjugated to the carboxyl

groups of G4.5-Gd2O3 nanoparticles to form PEG-G4.5-Gd2O3 conjugates via EDC/NHS coupling chemistry. For the synthesis of PEGylated, G4.5-Gd2O3 (10 mg, 0.4 µmol) was dissolved in 2 ml of ultra-distilled water and acidified with 1 N hydrochloride acid. Then, 9 mg of EDC and 5 mg of NHS were added and stirred for 14 h. NHS-activated G4.5-Gd2O3 was dialyzed, dried, and re-dissolved in sodium bicarbonate solution (pH 8.5). Next, O-(2-Aminoethyl) polyethylene glycol (HO-PEG-NH2) (0.1 g, 10 µmol) pre-dissolved in sodium bicarbonate solution (pH 8.5) was added to the G4.5-Gd2O3-NHS solution under continuous stirring. The mixture was stirred overnight to initiate the coupling chemistry. The resulting products were extensively subjected to dialysis using a cellular membrane with cut-off of 12–14 kDa against distilled water (1 l), and the outer phase was also replenished with 1 l of fresh distilled water every two hours. Then, the final solution was lyophilized overnight in a freeze dryer to obtain the final product. Characterization: The synthesized materials were confirmed using attenuated total reflectance (ATR) spectroscopy (JASCO FTIR-6700), and 1H nuclear magnetic resonance (NMR) spectra



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were recorded using a Bruker Avance 500.163 MHz NMR spectrometer with D2O as the solvent. For DOSY experiment, 30 mg of G4.5-Gd2O3-PEG NPs after purification (extensive dialysis using a cellular membrane with cut-off of 12–14 kDa against distilled water followed by lyophilization) was dissolved in D2O solvent in NMR tube. A superconducting quantum interference device (SQUID) was used to measure the magnetic properties of Gd2O3 nanoparticles. A high-resolution transmission electron microscope (HRTEM) (Philips Tecnai F20 G2 FEI-TEM) was used to analyze the morphology and size of Gd2O3 nanoparticles. The gadolinium content in the prepared G4.5-Gd2O3 nanoparticles was quantified by ICP-MS. X-ray photoelectron spectroscopy (XPS) measurements were carried out on a British VG Scientific ESCALAB 250 spectrometer fitted with XR5 Monochromatic X-ray Gun. T1 and T2 weighted and animal imaging were done with a Pharmascan 7T 16-cm bore horizontal MRI instrument. Briefly, Gd2O3 nanoparticles were serially diluted in distilled ionized ultra-filtered water in microcentrifuge tubes, as previously described.53 We have scanned the different concentration samples at 7T MRI machine using Bruker ParaVision Version 4.0 scanning software. Multi-slices multi-echoes (MSME) for T2-map and variable or multi-TRs for T1-map were used. Then we can get the T2 value (ms) from T2-map and T1 value (ms) from T1-map (just circle a ROI in the map center), and the analyze software (for fitting map and circle ROI) is mrvision version 1.6.6. 2.5.

MRI In Vitro Relaxivity Studies: The relaxivity value of Gd2O3 containing NPs

(G4.5-Gd2O3-PEG) was determined by measuring the longitudinal proton relaxation time T1 and T2 by 7T MRI as a function of gadolinium concentration, which was obtained using ICP-MS. Nanoparticles were dispersed in water at various concentrations (0.1, 0.05, 0.025, 0.0125, and 0.01 mM) at room temperature and were transferred to 200 µl Eppendorf tubes. 2.6.

In Vitro Cytotoxicity Assay: RAW 264.7 cells were cultured in 96-well plates at a

density of 1 x 105 cells per well and grown overnight in DMEM supplemented with 10% FBS. Then, the medium was replaced with fresh medium containing Gd-DTPA, G4.5-Gd2O3, or G4.5Gd2O3-PEG with different gadolinium concentrations and incubated for 24 h. Later, the medium with Gd-DTPA, G4.5-Gd2O3, or G4.5-Gd2O3-PEG were replaced with fresh medium containing 20 µl of an MTT (5 mg/mL) solution for 4 h. The medium with MTT was removed and replaced with 50 µl DMSO to dissolve the formazan crystals. Absorbance was read at a test wavelength of 450 nm and reference wavelength of 570 nm. Cell viability was calculated as:


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% = 2.7.

Absorbance of treated cells 100 Absorbance of control cells

Cell Apoptosis/Necrosis Analysis: RAW 264.7 cells were cultured into 6-well

dish with a density of 1 x 106 cells per well for 24 h and washed three times with PBS to remove dead cells. Then, Gd-DTPA, G4.5-Gd2O3, or G4.5-Gd2O3-PEG NPs were incubated with RAW 264.7 cells for 24 h. Then, the cells were washed, collected, and suspended in 500 µl of a 1x Annexin V-binding buffer. Alexa Fluor@ 488 Annexin V and Propidium Iodide (PI) were used according to the manufacturer’s recommendation. Samples were incubated in the dark for 15 min. An additional 400 µl of 1x Annexin V-binding buffer was added and mixed gently with the samples prior to analysis. 2.8. Gadolinium

Xylenol Orange Disodium Salt for Spectrophotometric Determination of Free Ions:

A

xylenol

orange

disodium

salt

(3,

3′-bis

[N,

N-

bis(carboxymethyl)aminomethyl]-o-ocresolsulfonephthalein tetrasodium salt) acetic acid buffer (pH 5.8) and calibration curve were obtained according to previously report.54 For determination of gadolinium ions, 50 µl of sample was added to 2 ml of xylenol orange disodium salt acetic acid buffer prior to UV detection at 573 nm and 433 nm. The amount of free gadolinium ions was calculated based on the fitted equation. 2.9.

Mimicking the Release of Gadolinium Ions

The leakage of Gd3+ from contrasting agents of G4.5-Gd2O3-PEG NPs in vivo conditions was mimicked by evaluation of the amount of Gd3+ release in phosphate buffer at 37°C under sink conditions (20 ml) by continuous stirring over a period of five days. To assess the amount of leaked gadolinium ions, spectroscopy quantification was performed using the xylenol orange indicator at 24-h intervals, based on a previously reported protocol.54 The absorbance of free Gd3+ leakage was measured at 433 and 578 nm. 2.10. In Vivo MRI: BALB/c female mice with weights of 19-21 g were purchased from BioLASCO Taiwan Co., Ltd. All mice experiments were done based on the guidelines of the Institutional Animal Care and Research Committee of the Taiwan Mouse Clinic (Academia Sinica, Taiwan). Briefly, the animals were anesthetized by isoflurane inhalation, and 3 mice were used in each group for in vivo MRI. Positive T1 and negative T2-weighted MRI imaging were performed according to the following parameters: inversion recovery times (TI) = 40, 60, 100,



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150, 300, 600, 1000, 2000, 3000, and 5500 ms, repetition time (TR) =500 ms, echo time (TE) = 10 ms, matrix size = 256 x 256, FOV = 50 x 50 mm, flip angle = 15, slice thickness = 1 mm, slice number = 10, number of requisition = 4, total scan time = 4 min 16 s and TR = 6000 ms, first TE = 10 ms, echo spacing = 10 ms, number of TE = 10, FOV = 6 x 6 cm2, matrix size = 256 x 256, slices thickness = 1.5 mm, NEX = 2, respectively. Magnetic nanomaterials (G4.5-Gd2O3-PEG) at a dose of 0.5 µmol Gd/kg body weight of BALB/c mice (n = 3) were injected via tail vein. Images of organs, including kidney, liver, spleen, and bladder, were acquired at time intervals of 0 min, 5 min, 30 min, 60 min, 90 min, and 24 h. 2.11.

Biodistribution Studies: Major organs were collected from sacrificed mice which

were injected with G4.5-Gd2O3-PEG nanoparticles for 24 h, 8 days, and 14 days. Then the organs were grounded, homogenized, treated with aqua regia, heated at 90ºC filtered and then the filtrates were analyzed by ICP-MS.

3. RESULTS AND DISCUSSION 3.1.

Synthesis and Characterization of G4.5-Gd2O3 Nanoparticles

A preparation mechanism was designed for the inclusion of biocompatible Gd2O3 nanoparticles in the interior of G4.5 PAMAM dendrimers. First, paramagnetic Gd3+ ions were encapsulated into three dimensional nanostructured PAMAM dendrimer cores. Then, the Gd3+ ions were oxidized with alkali to provide a Gd2O3 colloidal nanoparticle suspension in the interior of dendrimers, as shown in Scheme 1. For further characterization, the ratio of dendrimer to Gd3+ was fixed at a molar ratio of 1:5, since further increase in the content of Gd3+ resulted in precipitation and compromised the contrasting ability.


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O O

Acidify with 1N HCl

pH =3.0

GdCl3

O

EDC, NHS

N O

NaOH

(G4.5-NHS)

NH2-PEG-OH (10K)

PEG-OH NH O

O HN PEG-OH (10K)

MR Imaging

Scheme 1: Schematic diagram of the synthesis of G4.5-Gd2O3 and G4.5-Gd2O3-PEG followed by intravenous injection to normal mice for in vivo MRI. TEM images revealed uniform spherical structures with diameters of 3-5 nm, as shown in Figure 1A. The zeta potential of G4.5-Gd2O3 NPs was approximately -15.7 mV, which was higher than that of native G4.5 PAMAM dendrimers (-56.5 ± 0.2 mV).55-56 This can be attributed to the chelation of leaked gadolinium (Gd3+) ions with the -COOH surface of the dendrimers.57 In addition, we used DLS for size measurement, as shown in Table S2; here, the size was slightly higher than expected. This may be due to the aggregation formed from the leaked Gd3+ and the carboxylic surface of the dendrimer; this result concurs with the result of the leakage experiment.



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To further verify the structure of Gd2O3 NPs in the interior of PAMAM dendrimer, HRTEM and SEM were used. As predicted, dense Gd2O3 nanoparticles widely distributed at the core of the dendrimer, as illustrated in Figure 1B and Figure S1A, respectively. The HRTEM image lattice spacing was 0.28 nm, which corresponds to the (400) plane of Gd2O3 nanoparticles.58 Next, the chemical composition and crystal structure of the gadolinium oxide nanoparticles were determined by X-ray diffraction (XRD), as shown in Figure 1C. The result is consistent with the standard data of JCPDS 12-0797.59-60 A broad XRD pattern indicated that most of the ultra-small Gd2O3 nanoparticles were not fully crystallized due to their small particle diameter.24,

61

Energy dispersive spectroscopy (EDS) mapping revealed that the nanostructure

consisted of C, N, O, and Gd (Supporting Figure S1B and S1C, and Supporting Table S1), indicating that Gd2O3 was successfully grown on the core of PAMAM dendrimers. The gadolinium contents were also quantified in the complex by ICP-MS. The results indicated that approximately 10.5% gadolinium was present in the nanoparticles.


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B

A

0.28 nm

C o

After calcination at 400 C for 3 h

As prepared G4.5-Gd2O3

Intensity (a.u.)

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Gd2O3

20

30

40

50

60

2θ



Figure 1: (A) TEM indicating uniform distribution and spherical morphology of G4.5-Gd2O3 nanoparticles. (B) HRTEM image of Gd2O3 nanoparticles in the core of dendrimers. (C) XRD spectra of G4.5-Gd2O3 NPs before and after calcinations and Cubic Gd2O3 JCPDS 12-0797 data. The results of XPS, as presented in Figure 2, confirmed the presence of gadolinium in the form of Gd2O3. Gd consists of different binding energies with different spin orbits. The Gd 4d spectra, with a peak at 142.1 eV and Gd 3d5/2 peak at 1187 eV, confirmed the formation of Gd2O3 nanoparticles.26, 62 Figure 2A represents the O (1s) spectrum, with three distinct peaks at 536.3, 532.4, and 531.8 eV. The peak at 532.4 eV corresponds to the oxygen in the Gd2O3 nanoparticles 58

, whereas the peaks at 531.8 and 536.3 eV originate from the dendrimers. As shown in Figure

S2, the N (1s) spectrum contains a peak at 400.1 eV, which corresponds to the N atoms in the interior of dendrimers with Gd2O3 nanoparticles.

1000

A O (1s)

Intensity (a.u.)

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532.48

500

536.29 531.79

0 540

538

536

534

532

530

Binding energy (eV)


528

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1000

B Gd 3d5/2

800

Intensity (a.u.)

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600

400

200

0 1192

1188

1184

1180

Binding energy (eV)

Figure 2: X-ray photoelectron spectrum (XPS) of (A) O (1s) and (B) Gd (3d5/2) for G4.5-Gd2O3 nanoparticles. The magnetic property of G4.5-Gd2O3 nanoparticles was measured using superconducting quantum interference device (SQUID) magnetometer at 5 and 300 K. The mass-corrected zerofield cooled (ZFC) M versus the applied field (H) i.e., M-H curves (−5≤H≤5 tesla) at T=5 and 300 K are shown in Figure 3A, and magnetization (M) (emu/g) versus T curves ZFC M-T curve (5 ≤ T ≤ 300 K) at H = 3000 Oe, which are shown in Figure 3B. Saturation magnetization was calculated to be 0.02 emu at 5 K. Moreover, the M-H curves at T = 5 and 300 K indicate that both coercivity and remanence were zero. This demonstrates that Gd2O3 nanoparticles were paramagnetic as low as T = 3K, which is consistent with previous reports.53, 63 Thus, ultra-small Gd2O3 containing nanoparticles could efficiently induce the relaxation of water protons. A high r1 value was observed from the phantom image.



A 0.002

0.001

0.000

0

50

100

150

200

250

300

Temperature (K)

at 5 K at 300 K

M(emu)

M (emu)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

-60000

-40000

B 0.02

-20000

0.00

0

20000

40000

60000

-0.02

H(Oe)


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Figure 3: (A) Mass corrected temperature-dependent magnetization curve (M-T) of Gd2O3 nanoparticles at H of 3000 Oe. (B) Field-dependent magnetization (M-H) curve of Gd2O3 nanoparticles at 5 and 300 K. 3.2. Grafting of G4.5-Gd2O3 Nanoparticles with Polyethylene glycol Herein, G4.5-Gd2O3-PEG nanoparticles were prepared by EDC/NHS coupling chemistry of the functionalized G4.5-Gd2O3 and the amine terminated PEG groups. The colloidal stability of G4.5Gd2O3-PEG was studied by measuring the changes of hydrodynamic diameter. The result indicates that the hydrodynamic diameter of the NPs was approximately 50.4 nm, as illustrated in Table S2. The net surface charge of nanoparticles was ~ -0.2 mV (Table S2), which implies that the surface of the dendrimers was covered with PEG. Although the surface charge of the nanoparticles tended to 0 mV, PEGylation G4.5-Gd2O3 nanoparticles still exhibited good colloidal stability for a month. This is due to the PEG hydrophilic shell on the surface of particles that prevents further aggregation.64 We have confirmed that there was no precipitation of the G4.5-Gd2O3-PEG solution, and the particle sizes remained unchanged. As shown in Figure 4A, ATR-FTIR revealed the presence of C-H at 2850-2940 cm-1, C=O at 1580-1650 cm-1, and C-O at 1060-1110 cm-1, indicating the conjugation of PEG. The characteristic peak below 900 cm-1 is due to the Gd-O stretching in Gd2O3 nanoparticles.65 The number of PEG per G4.5 PAMAM dendrimer was assessed by 1H-NMR diffusion spectra (2D DOSY) were acquired as a function of the gradient strength, as shown in Figure 4B-4D. The chemical shifts at δ 3.75 ppm correspond to the PEG protons and the shifts at 2.54 to 3.68 ppm correspond to multiple protons of G4.5 PAMAM.56, 66 Figure 4B shows the results of 2D DOSY experiments for G4.5-Gd2O3-PEG nanoparticles. The DOSY map (Figure 4C) of 1H-NMR signals belonging to PEG and dendrimer complexes correlate with the one diffusion coefficient of 5.5x10-7 m2s-1. From 2D DOSY spectra (Figure 4D), the 1H-NMR was extracted, followed by peak integration to obtain a ratio of PEG to dendrimer of ~ 22:1 in the complex. Overall, the results show that PEG was successfully attached to the G4.5 PAMAM dendrimer.



A

G4.5-Gd2O3-PEG

G4.5-Gd2O3

G4.5-PEG

%T

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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PEG

G4.5

3500

3000

2500

2000

1500 -1

Wavenumber (cm )

B

D 2O

PEG Dendrimer

Acetone


1000

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


PEG D 2O

Acetone

C Dendrimer



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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D

Figure 4: (A) ATR-FTIR absorption spectra of powder samples and (B) PFA-NMR spectrum of as a function of gradient strength (C) Automated Bayesian DOSY transformed spectra (D) Extracted 1H NMR spectra from the DOSY spectrum of G4.5- Gd2O3-PEG nanoparticles. 3.3. Toxicity Studies of Nanoparticles To assess the amount of leaked gadolinium ions, spectroscopy quantification was performed using xylenol orange indicator. The acetic buffer solution of xylenol orange showed two absorption bands at 433 and 578 nm in the visible region (Figure 5A). The free Gd3+ concentration was proportional to the ratio of the absorbance at 578 and 433 nm (Figure 5B). Accordingly, the leaching of Gd3+ from G4.5-Gd2O3 and G4.5-Gd2O3-PEG nanoparticles were approximately calculated to be 2.17 and 0.123 µM, respectively from the calibration curve. The results indicate that 21.7% gadolinium ions were leaked from G4.5-Gd2O3 NPs, while leakage was reduced to a negligible level in the case of G4.5-Gd2O3-PEG (0.1%) nanoparticles. Further, mimicking the leakage of Gd3+ from contrasting agent (G4.5-Gd2O3-PEG) in vivo was performed by evaluating Gd3+ release from NPs in phosphate buffer at 37°C under sink conditions over five days. The


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percentage of free Gd3+ leakage for the first 24 h decreased and then equilibrated in the NPs within five days (Figure S3). This result indicates that there was no increase in free Gd3+ leakage from the synthesized G4.5-Gd2O3-PEG NPs in vivo.

[Gd3+] =0 3+ [Gd ] =0.125 µM [Gd3+] =0.25 µM [Gd3+] =0.5 µM [Gd3+] =1 µM [Gd3+] =2 µM G4.5-Gd2O3 G4.5-Gd2O3-PEG

A

Absorbance

0.4

0.2

0.0 300

400

500

600

700

Wavelength (nm)

0.08

B

Absorbance at 578/433 nm

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0.06

0.04

R2 = 0.93 y = 0.00482x + 0.03458 0.02

0.00 0.0

0.5

1.0

1.5

2.0

3+

[Gd ] (µΜ)


2.5


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Figure 5: Spectroscopic determination of Gd3+ leaching by xylenol orange dye: (A) absorbance vs wavelength, and (B) the ratio of absorbance at 578 and 433 nm vs concentration, the calibration curve obtained from standard free Gd3+ (Black square). The cytotoxicity of Gd-DTPA, G4.5-Gd2O3, and G4.5-Gd2O3-PEG nanoparticles on RAW 264.7 cells was tested with various gadolinium concentrations (Figure 6). The MTT result reveals that the G4.5-Gd2O3-PEG nanoparticles improved the viability of cells, as more than 96% cells with 0.1 mM of Gd3+ with were alive. However, before grafting of the nanoparticle with PEG, approximately 18% of normal cells were killed at 0.1 mM Gd3+ for G4.5-Gd2O3 nanoparticles, which can be attributed to the leaching of free gadolinium ions in G4.5-Gd2O3. Necrosis and apoptosis mechanisms of RAW 264.7 cells were assayed by flow cytometry. When the cell membrane is disturbed, phosphatidylserine could be translocated from the inner to the outer of the plasma membrane. This can be monitored by binding with annexin-V.67 In addition, the ability of PI internalize to the cell is dependent on the permeability/integrity of the cell membrane.68 In late apoptosis and necrosis, the plasma membrane loses its permeability and allows PI to intercalate into the nucleus. As shown in Figure 7, G4.5-Gd2O3-PEG (0.4%) induced less cell death than commercial Gd-DTPA (8.2%). On the other hand, the toxicity of G4.5-Gd2O3 (20.4%) can be attributed to leached gadolinium ions, which agrees with the MTT and xylenol orange tests. Thus, in vitro cell tests show that G4.5-Gd2O3-PEG nanoparticles are safe and suitable for further in vivo study.


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120

100

80

Cell Viability

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


60 Gd-DTPA G4.5-Gd2O3 G4.5-Gd2O3-PEG

40

20

0 0

20

40

60

80 3+

Concentration of Gd

100

(µΜ

Figure 6: Cell viability of RAW 264.7 cells incubated with Gd-DTPA, G4.5-Gd2O3 or G4.5Gd2O3-PEG nanoparticles at various Gd3+ concentrations for 24 h.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Figure 7: Flow cytometric analysis of apoptotic/necrotic RAW 264.7 cell populations stained with Annexin V-Alexa Fluor 488 and PI after incubation with (A) PBS as control, (B) Gd-DTPA, (C) G4.5-Gd2O3, and (D) G4.5-Gd2O3-PEG nanoparticles. (Q1: Annexin-V-Alexa Fluor 488-positive and PI-negative indicate early apoptosis; Q2: Both Annexin -V-Alexa Fluor 488- and PI-positive cells indicate late apoptosis. Q3: Both Annexin -V-Alexa Fluor 488- and PI-negative for viable cells; and Q4: Annexin-V-Alexa Fluor 488-negative and PI-positive indicates cell necrosis). 3.4. Phantom MR Imaging and Relaxivity Analysis The majority of T1 MRI contrast agents are based on gadolinium (Gd3+) chelates, since Gd3+ (4f7:8S7/2) has the largest seven unpaired electron.5,

69

In addition, the spin relaxation is

exclusively induced by the electron spin magnetic moment of the Gd3+-based contrasting agent. Herein, the contrast enhancement potential of G4.5-Gd2O3-PEG nanoparticles at various


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concentrations (0.01 mM–0.1 mM) was tested via T1 and T2 phantom in vitro using 7T MR imaging. As shown in Figure 8A, the contrast intensity of G4.5-Gd2O3-PEG NPs was enhanced due to increased relaxation rate of the surrounding water protons, which led to higher perturbation energy, shortened relaxation time (T1), and enhanced contrasting intensity, which is in agreement with previously reported results.34 Furthermore, when compared with clinically used Gd-DTPA,69 the nanoparticles showed contrast enhancement. Furthermore, for quantitative comparison of the r1 and r2 of gadolinium nanoparticles as a function of gadolinium ion concentration, the relaxation time (T1/T2) of each sample was determined. Figure 8B depicts the relaxivity rate as a function of gadolinium concentration. The r1 and r2 values were estimated from the slope of the plots of the inverse longitudinal (T1) and transverse (T2) relaxation times (i.e., 1/T1 and 1/T2). The relaxivity of (r1 and r2) values of G4.5Gd2O3-PEG nanoparticles were 53.9 and 182.8 mM-1s-1, respectively. The solid lines represent the linear fit or linear regression (R2) of experimentally obtained relaxation rates. In general, these results show that G4.5-Gd2O3-PEG nanoparticles are potential T1 and T2 MRI contrast agents. At present, the most commonly used T1 contrasting agents for clinical MRI investigations are gadolinium chelates such as Gd-DOTA and Gd-DTPA, which were reported to have T1 relaxivity values (r1) ranging from 3 to 5 mM−1 s−1. Notably, the r1 and r2 values were the highest after PEG was grafted onto G4.5-Gd2O3 nanoparticles, as shown in Table 1. These values were also significantly higher than those of currently available gadolinium based T1 contrasting agents. Thus, the introduction of hydrophilic PEG could have shortened the relaxation time and enhanced the magnetic signal intensity by ensuring that Gd2O3 nanoparticles interacted with surrounding water protons.70 Furthermore, the outermost shell electron of gadolinium in Gd2O3 NPs offered seven unpaired electrons for water hydration to induce longitudinal relaxation of water protons.22, 25 The decoration of nanocarriers with dendrimers provided increased solubility, while incorporation of PEG resulted in suitable hydrophilic function, stability, and prolonged circulation of these systems in vivo.71



A

B 20

Relaxivity rate (1/T)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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15

r1 r2

R2 = 0.9931 r2 = 182.81 s-1mM-1

10

R2=0.9965 r1 = 53.92s-1mM-1

5

0 0.00

0.02

0.04

0.06

0.08

0.10

Concentration of Gd3+(mM) Figure 8: (A) T1-T2 weighted phantom MR imaging of G4.5-Gd2O3-PEG NPs at different Gd3+ concentrations and (B) variation of experimentally measured longitudinal relaxivity, r1 (1/T1) and r2 (1/T2) for G4.5-Gd2O3-PEG NPs as a function of Gd3+ concentration.


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Table 1. Relaxivity values (r1 and r2) of related Gd-containing nanoparticles Samples

H (tesla)

T (ºC)

r1 (s-1mM-1)

r2 (s-1mM-1)

References

Gd-DTPA

0.47

25

3.8

4.2

69

Gd-DOTA

0.47

25

4.2

4.6

69

Gd2O3-IC

1.5

22

28

52

72

Gd2O3-PVP

7

25

12.1

33.2

34

Gd2O3-SiPEG

7

25

4.4

28.9

27

Gd2O3 (Cubic like)

3

25

19.5

89.4

35

Gd2O3 (Sphere)

3

25

22.2

128.9

35

G4.5-Gd2O3-PEG

7

25

53.9

182.8

This work

3.5. T1-T2 Weighted In Vivo MR Images Coronal T1-T2 weighted MRI images with spin-echo sequence were acquired after the synthesized materials were delivered into BALB/c mice. G4.5-Gd2O3-PEG nanoparticles with the largest r1 and r2 value were used to obtain the in vivo T1/T2 MR images of mice at an MR field of 7T system. After intravenous tail vein injection (100 µl, 0.5 µmol per kg body weight) of G4.5-Gd2O3-PEG nanoparticles, images were taken at 0 min (pre-injection) and 1.5 h (postinjection) at different organ positions (Figure 9). Figure 9A shows that the nanoparticles were found throughout the body in the reticuloendothelial system (RES) postinjection. Since G4.5-Gd2O3-PEG was demonstrated to have a large hydrodynamic particle size, typically 50 nm, it could be trapped by RES absorption and accumulate in the liver and spleen.73 On the other hand, positive contrast enhancement was observed as the nanoparticles were taken up by respective organs after 5 min, 30 min, 60 min, and 90 min postinjection. As shown in Figure S4A, bright signal intensity, which later declined, was observed in the small intestine 30 min postinjection. This may be attributed to the enterohepatic circulation (EHC) of G4.5-Gd2O3-PEG contrasting agents. Similarly, bright signals were observed in the kidney postinjection and declined after 1 h. In contrast, signal intensity in the liver was slightly enhanced throughout compared to in the baseline control. On the other hand, the intensity in the bladder was enhanced 1 h postinjection, implying that the kidney removed nanoparticles from the vascular compartment to the gall bladder. The



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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results of the signal to noise (S/N) ratio of the regions of interest (ROIs), as shown in Figure 9B, are in accordance with the imaging findings. Generally, excretion was slower in the major organs, such as liver, kidney, spleen, and bladder, and was influenced by the slight negative charge and hydrodynamics size of G4.5-Gd2O3-PEG NPs. Similarly, the T2-weighted MRI images reveal definite negative contrast enhancement for G4.5-Gd2O3-PEG nanoparticles due to their higher r2 value. After injection, slight darkness in the kidney was observed (Figure 10A). After 24 hours, the darker contrast enhancement in the kidney indicated that the nanoparticles were removed out by the kidney. But, there is no significant intensity change in the liver (Figure S4B). The S/N ratio results (Figure 10B) indicate that the particles circulated to the liver slowly and the S/N at the liver was lower than that at the kidney. This is in accordance with the T2-weighted MRI images. In general, these novel Gd-containing nanoparticles were shown to be effective dual modal (T1-positive and T2-negative) contrasting agents by both in vitro phantom and in vivo images. In addition, 2 weeks after performing MRI in vivo, mice remained healthy without losing weight. Furthermore, the main organs were extracted for ICP-MS analysis of gadolinium content (Figure 11). ICP-MS analysis shows that the majority of G4.5-Gd2O3-PEG nanoparticles were located in the kidney, which is consistent with in vivo image results. The concentrations of gadolinium content in the kidney rapidly decreased after 24 h, but decreased gradually in the spleen and liver.

A 5 min

Pre-injection

30 min

I

K


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


60 min

90 min

24 h

B Kidney 160

Intestine Liver

120

S/N

80

40

pre

post-5mins post-30mins

post-1hr


post-1.5hr

post-24hr


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Figure 9: Coronal T1-weighted MRI images of mouse after intravenous injection of G4.5-Gd2O3PEG NPs at 0 min, 5 min, 30 min, 60 min, 90 min, and 24 h: (A) kidney (K) and large intestine (I) and (B) signal to noise ratio (SNR) of the regions of interest (ROIs) with respect to injection time. Error bars were based on triplicate measurements. A Pre-injection

60 min

5 min

90 min

30 min

24 h


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B 120.00

Kidney Liver

100.00 80.00 S/N

60.00 40.00 20.00 0.00 pre

post-5mins post-30mins

post-1hr

post-1.5hr

post-24hr

Figure 10: Coronal T2-weighted MRI images of mouse after intravenous injection of G4.5-Gd2O3PEG NPs at 0 min, 5 min, 30 min, 60 min, 90 min, and 24 h: (A) kidney (K) and (B) signal to noise ratio of the regions of interest of different organs with respect to injection time. Error bars were based on triplicate measurements.

Concentration of Gd3+ in G4.5-Gd2O3PEG (ng/mL)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1800

Spleen

1600

Kidney

1400

Liver

1200 1000 800 600 400 200 0 1

8 Time (dayes)


14


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Figure 11: Biodistribution of gadolinium content in the organs of sacrificed mice with intravenous injection of G4.5-Gd2O3-PEG nanoparticles (0.5 µmol/kg body weight) for 1, 8 and 14 days. Error bars were based on triplicate measurements. 4. CONCLUSIONS In this study, Gd2O3-based nanoparticles with high biocompatibility and contrasting ability were synthesized. Gd2O3 nanoparticles were grown in the core of G4.5 PAMAM dendrimers using alkaline solution and grafted with PEG to enhance the biocompatibility and contrasting capability in vitro and in vivo. The longitudinal relaxation rate r1 (1/T1) was calculated to be 53.9 mM-1s-1, which is approximately five times superior to that of clinically used contrasting agents. Due to the unique magnetic properties of gadolinium and the dense population of gadolinium per G4.5Gd2O3-PEG nanoparticles, the prepared nanoparticles showed superior T1-T2 weighted phantom MRI imaging ability compared to clinically used contrasting agents (Gd-DTPA or Gd-DOTA). Results from in vivo MRI analysis indicate that the prepared material is an ideal candidate dual MRI image. The designed contrasting agent was more powerful, with respect to MRI signal, when compared to the agents clinically available today. ACKNOWLEDGMENTS The authors would like to thank the Ministry of Science and Technology, Taiwan (MOST 1052221-E-011-133-MY3 and 105-2221-E-011-151-MY3) for providing financial support and Taiwan Mouse Clinic (MOST 104-2325-B-001-011) which is funded by the National Research Program for Biopharmaceuticals (NRPB) at the Ministry of Science and Technology (MOST) of Taiwan for technical support in animal experiments. ASSOCIATED CONTENTS Supporting Information Scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX) spectrum, Xray photoelectron spectroscopy (XPS) analysis of G4.5-Gd2O3 NPs, DLS, and Zeta potential of G4.5-Gd2O3-PEG nanoparticles at 25°C, percentage of free Gd3+ leakage in the G4.5-Gd2O3-PEG NPs vs time, coronal T1-weighted MRI images of mice after intravenous injection of G4.5-Gd2O3-


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Table of Content (TOC)

H -O G PE

H N

O

HR-TEM

O

N H

H ) 0K (1

IV Injection

-O G PE

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


T1 MR imaging


Encapsulation of Gadolinium Oxide Nanoparticle (Gd2O3) Contrasting

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