Synthesis of PEG-Iodine-Capped Gold Nanoparticles and Their Contrast Enhancement in InVitro and InVivo for X-Ray / CT

1 Department of Nuclear Medicine, Chonbuk National University Hospital, Dukjin-gu, Jeonju, Geumam-dong 634-18, Republic of Korea 2 Cyclotron Research Center, Chonbuk National University Hospital, Dukjin-gu, Jeonju, Geumam-dong 634-18, Republic of Korea 3 Research Institute of Clinical Medicine, Chonbuk National University Medical School and Hospital, Dukjin-gu, Jeonju, Geumam-dong 634-18, Republic of Korea


Introduction
X-ray computed tomography (CT) has been widely used as an imaging tool for noninvasive diagnosis of many diseases.Water-soluble iodinated ionic and nonionic small molecules are commonly used as CT contrast agents more than 10 years [1][2][3][4][5][6].However, CT contrast agents containing iodine have got disadvantages, in which contrast agents are excreted from the body very rapidly, nonspecific, and have renal toxicity.Various contrast agents based on nanoparticles have recently been developed to overcome the short circulation time of iodinated contrast agents.Gold nanoparticles are an example of such CT contrast agent.It is well known that gold has a higher atomic number ( 79 Au versus 53 I) and a greater absorption coefficient than iodine (k edge; Au, 80.7 keV versus I, 33 keV) [7,8].During the past few years, biocompatible polymer-capped nanoparticles or water-soluble macromolecular nanoparticles have been investigated for suitability as CT contrast agents in nanomaterials and nanomedicine [9][10][11][12][13].Gold nanoparticles have been used for the detection of target materials by exploiting the optical properties of aggregation of gold with the desired substrates [14][15][16][17][18][19][20].These nanoparticles with different targeting ligands can be used as drugs and for gene delivery into cells in various diagnostic and therapeutic research settings [21][22][23][24][25][26][27].Gold and iodine atoms have an additional interesting feature: halide ions and organic compounds absorb onto the transition-metal surfaces through mutual interactions [28][29][30][31][32].This absorption can result in more stable complexes.
The preparation of gold nanoparticles through the citrate reduction method generally increased the stability of colloidal gold nanoparticles via the absorption of citrate ions onto the surfaces of the gold nanoparticles [33,34].The absorbed materials are linked to the surfaces and form a layer of absorbed molecules.For example, self-assembled monolayer (SAM) products of gold nanoparticles are often formed via chemisorptions of the thiol (-SH) functional group by the gold surface [35][36][37][38][39]. Halide ions and various organic or inorganic compounds can be absorbed by the metal surfaces.Chemisorption is one class of absorption that is driven by a chemical reaction that creates strong new bonds at the exposed metal surfaces.Because the nature of chemisorptions differs from system to system, the chemisorptions of a metal depend on its chemical identity and surface structure.
Gold halide is well known to have a peculiar electron system, and halide ions are known to have different affinities to gold surfaces [40].The relative binding strengths of gold halides are I > Br > Cl [41,42].The addition of iodide ions to gold nanoparticles leads to aggregation and fusion of gold nanoparticles [43][44][45].This suggests that gold nanoparticles with iodide are capable of not only being displaced but are also stabilized by the organic or inorganic materials.
In this study, we report the preparation of gold nanoparticles with iodine using chemisorptions of iodine onto the surface of gold, resulting in a CT contrast agent that can be used for in vivo blood-pool imaging.

Preparation of the Colloidal Gold Nanoparticles.
The colloidal gold nanoparticles were prepared using the following method [10].Gold chloride trihydrate (0.050 g; 1.27 × 10 −4 mole) was dissolved in 500 mL deionized water.Sodium citrate tribasic dihydrate solution (17.5 mL of a 1% solution) was added to the refluxed gold chloride solution as a reducing agent, followed by reflux and vigorous stirring for another 15 min.Sodium citrate solution caused an instant color change of the mixture to wine-red.The prepared colloidal gold nanoparticles had a 10 nm core diameter.

Preparation of the Methoxy PEG-Capped
AuNPs.A 0.105 g (2.10 × 10 −5 mole) of methoxy PEG sulfhydryl 5000 solution was dissolved in 20 mL deionized water and added to the prepared colloidal gold nanoparticles, followed by gentle stirring for 12 h at room temperature.The reaction solution was collected using a centrifugal filter (molecular weight cut off: 50 kDa, Millipore Corporation, Billerica, MA, USA) and washed three times with deionized water.

Preparation of the PEG-Iodine-Capped
AuNPs.Sodium iodide solution (0.01 M; 25 mL) was combined with the prepared citrate ion-capped gold nanoparticles and was gently stirred at room temperature for 1 h.Methoxy PEG sulfhydryl 5000 solution was then added to the iodinecapped gold nanoparticles solution and stirred gently for 12 h at room temperature.The final solution was concentrated using a centrifugal filter and washed three times with deionized water.The condensed iodine-containing products were stabilized with methoxy PEG sulfhydryl.

Characterization of the Methoxy PEG-Capped AuNPs and the Methoxy PEG-Iodine-Capped AuNPs.
The absorption spectra of the methoxy PEG-capped AuNPs and the methoxy PEG-iodine-capped AuNPs were assessed using a UV-visible spectrophotometer (HP 8453, Hewlett Packed, Germany).The surface compositional atoms of the gold nanoparticles were characterized using X-ray photoelectron spectroscopy (XPS) (AXIS-NOVA, Kratos, MC, UK).The X-ray source was monochromatic A1Kσ, 1486.6 eV.The sizes and shapes of the methoxy PEG-capped AuNPs and the methoxy PEGiodine-capped AuNPs were observed with transmission electron microscope (TEM) (H-7650; Hitachi, Tokyo, Japan).The samples were prepared commercially onto copper grids bearing formvar carbon film (FCF200-Cu: Electron Microscopy Science, Hatfield, PA, USA).The hydrodynamic particles sizes of the methoxy PEG-capped AuNPs and the methoxy PEG-iodine-capped AuNPs in water were measured using dynamic light scattering (DLS) (Microtrac-UP150; Microtrac, Largo, FL, USA).The concentrations of gold in the AuNPs were measured using an inductively coupled plasma mass spectrometer (ICP-MS) (Agilent 7500a; Agilent Technologies, Santa Clara, CA, USA).

Static Image of Methoxy PEG-125 Iodine-Capped Gold
Nanoparticles.The colloidal gold nanoparticles were prepared using citrate reduction method and a preparation of methoxy PEG-125 Iodine-capped (660 μCi, 38 ng) gold nanoparticles were then progressed using a radioactive isotope to confirm an existence of iodine on the surface of gold nanoparticles.Iodine-125 gamma camera imaging and imaging processing were performed using a smallanimal imaging system with pinhole collimation (aperture diameter = 1 mm; focal length = 9 cm) and a 15-to 45-keV photopeak energy window (X-SPECT/CT, GE Healthcare, Uppsala, Sweden).

In Vitro
Experiments.An in vitro experiment was performed to compare the degree of contrast enhancements of the methoxy PEG-capped AuNPs and the methoxy PEGiodine-capped AuNPs at the same concentration of gold and to determine the relative CT attenuation of the methoxy PEG-iodine AuNPs compared with that of the commercial iodinated contrast agent (Optiray TM 320, Tyco Healthcare, Canada).Optiray320 was prepared with serially diluted samples in distilled water.The serial samples contained the following amounts of iodine per milliliter:  First, the initial Au 0 atoms generated using the citrate reduction method were assembled to form stabilized citrate ion-capped AuNPs.Second, the colloidal gold nanoparticles were reacted with iodide ions in sodium iodide solution.Finally, methoxy PEG-SH was added to the iodine-capped AuNPs solution, and AuNPs was stabilized by methoxy PEG-SH.6.30 × 10 −5 mole; (e) water (control).The concentration of gold was determined by ICP-MS to be 3.70 × 10 −5 mole.All samples were placed in 200 μL PCR tubes.The CT images were acquired using a CT scanner (X-SPECT/CT, GE Healthcare, Uppsala, Sweden) with an estimated X-ray power of 75 kVp.The final reconstructed images were converted to digital images with the AMIRA software 3.1 (San Diego, CA, USA).

Acquisition of CT Imaging in Mice.
All animal experiments were performed in compliance with the policies and procedures of the Institutional Animal Care and Use Committee for animal treatment of Chonbuk National University.A female balb/c mouse (6 wks, weighting 25 g) was purchased from Orient Bio Inc., (Seongnam, South Korea) and was used in the animal study.The degree of contrast enhancement of the methoxy PEG-iodine-capped AuNPs was assessed in vivo.The mouse was anesthetized in an induction chamber with 2% isoflurane in oxygen and was maintained in 1.5% isoflurane in oxygen during CT imaging.The methoxy PEG-iodine-capped AuNPs were injected into the mouse via a catheter in the tail vein.The images were acquired at 30 m, 60 m, 1, 2, 6, 12, 24, 48, and 120 h after postinjection.The CT images were acquired using X-SPECT/CT with an estimated X-ray power of 75 kVp.The final reconstructed images were converted to digital images with the AMIRA software 3.1.

Results and Discussion
The design of the procedure for the preparation of methoxy polyethylene glycol (PEG)-iodine-capped gold nanoparticles (AuNPs) is shown in Figure 1.The methoxy PEG-capped AuNPs and the methoxy PEG-iodine-capped AuNPs were prepared according to Figure 1 and were observed to be deep red-wine-colored solutions.Figure 2 showed characteristic surface plasmon resonance (SPR) bands of methoxy PEGcapped AuNPs and methoxy PEG-iodine-capped AuNPs at the different wavelengths 520 and 521 nm.The SPR bands of the gold nanoparticles showed red shifts from 518 nm to 520 and 521 nm due to chemisorption between the sulfhydryl (-SH) group and the iodine on the surfaces of the gold nanoparticles.The chemisorption of PEG-SH on the surfaces of gold nanoparticles contributed to the stabilization of these AuNP (518 nm) AuNP @ PEG (520 nm) AuNP @ I-PEG (521 nm) gold nanoparticles.The substitution of PEG-SH onto gold nanoparticles resulted in the release of citrate ions from the surfaces of the gold nanoparticles.The chemisorption of iodine also induced changes in the surface charges of the gold nanoparticles, resulting in the assembly of gold nanoparticles.As a result, the red shifts of the SPR bands of gold nanoparticles were observed in UV-visible absorption spectra.
The sizes and shapes of the prepared gold nanoparticles were confirmed using transmission electron microscopy (TEM) and dynamic light scattering (DLS) as shown in Figures 3 and 4. The methoxy PEG-capped AuNPs had an average core diameter of 10 nm and were dispersed uniformly, but the methoxy PEG-iodine-capped AuNPs were slightly aggregated due to the addition of iodide ion during the preparation procedure (Figure 3(d)).The adsorbent iodide ions in aqueous sodium iodide induced the aggregation of gold nanoparticles [43].The hydrodynamic size distributions of the methoxy PEG-capped AuNPs and the methoxy PEG-iodine-capped AuNPs in water were measured using DLS.The sizes of the AuNPs were confirmed as   a,b,c The binding energies for Au and Au-I are from [29].
40 nm and 41 nm, respectively (Figure 4).The surface stress of gold nanoparticles was increased due to the mismatch between iodine and gold, leading to the slight aggregation of gold nanoparticles [44].Because iodine was added to gold nanoparticles within a short time period (one hour), sodium iodide prevented the severe aggregation of gold nanoparticles, and then the methoxy PEG-iodine-capped AuNPs were stabilized via capping with methoxy PEG-SH.The components of the methoxy PEG-iodine-capped AuNPs and the methoxy PEG-capped AuNPs were investigated using X-ray photoelectron spectroscopy (XPS).The metal-halide coordinated complex is thought to involve a strong bond.Iodide ions underwent spontaneous oxidation to generate neutral iodine atoms, zero-valent iodine, in aqueous sodium iodide [28,43].The newly formed iodine could be absorbed onto the surfaces of gold nanoparticles by virtue of their strong affinity.Iodine replaced citrate ions on the gold nanoparticles through chemisorption.Binding energy values for gold and iodine were investigated in Figure 5.The Au 4f 7/2 and 4f 5/2 spectra of the methoxy PEG-iodine-capped AuNPs (sample 2) were obtained at 84.1 eV and 87.8 eV, respectively.The binding energy of Au 4f 7/2 was changed slightly from 83.8 eV to 84.1 eV through treatment with iodine.According to a previous report, the characteristic binding energy of Au 4f 7/2 , gold (Au) is 83.8 eV if untreated with iodine and that of Au 4f 7/2 in a gold-iodine interface is 83.9 ± 0.5 eV (Table 1) [29].After the chemisorption of iodine to gold nanoparticles, the binding energy of gold in nanoparticles increased slightly as expected from the change in the level of gold-iodine complex described in the literature.The binding energy of Au 4f 7/2 in the methoxy PEG-capped AuNPs (sample 1) also changed from 83.8 eV to 84.0 eV.This shifted binding energy was ascribed to the AuNP-PEG assemblies representing the bonded thiol (-SH) of PEG-SH with gold nanoparticles.These results indicate that the changes of binding energy of the methoxy PEG-capped AuNPs and methoxy PEG-iodinecapped AuNPs was resulted by chemisorption via thiol (-SH) and iodine.Iodine, 3d 5/2 and 3d 3/2 spectra were clearly visible in the methoxy PEG-iodine-capped AuNPs due to the formation of gold-iodine bonds at 619.3 eV and 630.8 eV.However, the energy of iodine 3d 5/2 was not clearly observed in the methoxy PEG-capped AuNPs.The binding energy of iodine in gold-iodide is known to be 618.3 ± 0.5 [28].After the addition reaction of iodine, the band of iodine 3d 5/2 was shifted higher, from 618.3 eV to 619.3 eV.In this study, the iodine species that were generated were established as iodine (I 2 ) molecules.These results indicate that there was no gold-iodide (Au-I) complex with inserted iodide ions (I − ).Judging from the these results, we suggest that the binding of neutral iodine (I 2 ) molecules to gold nanoparticles is more favorable than the combination of iodide ions (I − )  Therefore, with the analysis of acquired images of tube samples, we could derive the amount of iodide, although we did not check its iodide exactly, in methoxy PEG-iodinecapped AuNPs to above 6.30× 10 −5 mole.When comparing by atom concentration additionally, methoxy PEG-iodinecapped AuNPs was 1.7 times lower than Optiray320, but contrast intensity has shown almost the same signal.We observed obvious contrast enhancement at the heart, aorta, liver, and kidney after injection compared with that of the untreated control as shown in Figure 8.The contrast enhancement was shown for 24 h in the aorta.We found that methoxy PEG-iodine-capped AuNPs cleared from the heart after 48 h after injection and were taken up by the reticuloendothelial system (RES) system in the liver.The renal artery and renal pelvis were also observed vividly.The degree of contrast enhancement of methoxy PEGiodine-capped AuNPs was maintained from immediately up to 5 days after injection at the same position.From the above results, we confirmed that the methoxy PEG-iodinecapped AuNPs prolonged a significant blood circulation time compared with the commercialized contrast agent with iodine.The CT images were displayed in sequence according to the time-dependence in Figure 8.There was no evidence of apparent toxicity for the duration of the enhancement.

Conclusions
In this study, we described the preparation of new concept of PEG-iodine capped gold nanoparticles using the chemisorption mechanism of iodine atoms onto the surface of gold nanoparticles, and this was stabilized with methoxy PEG-SH.The additional method of iodine capping on the gold nanoparticles brought about better enhancement for X-ray compared to iodine non-capped gold nanoparticle, and the quality of X-ray/CT imaging of polymer-capped gold-iodine complex was more improved compared to Optiray320 in spite of using smaller doses.One of the problems for using the conventional contrast agents in X-ray imaging is that large amounts of the current polyiodinated contrast agents are required to obtain the appropriate enhancement and the other short blood circulation time of them.New concept of iodine capping onto gold nanoparticles would be a good candidate for solving these problems.

Figure 1 :
Figure1: Synthetic scheme of methoxy PEG-iodine-capped AuNPs.First, the initial Au 0 atoms generated using the citrate reduction method were assembled to form stabilized citrate ion-capped AuNPs.Second, the colloidal gold nanoparticles were reacted with iodide ions in sodium iodide solution.Finally, methoxy PEG-SH was added to the iodine-capped AuNPs solution, and AuNPs was stabilized by methoxy PEG-SH.

Figure 5 :
Figure 5: X-ray photoelectron spectroscopic (XPS) data for the methoxy PEG-capped AuNPs (sample-1) and the methoxy PEG-iodinecapped AuNPs (sample-2).These data show the change of binding energy of gold and iodine.

Figure 6 :
Figure 6: The methoxy PEG-125 iodine-capped gold nanoparticles showed high radioactive through the static image.Photographic image of methoxy PEG-12 iodine-capped gold nanoparticles (a) and static image of methoxy PEG-125 iodine-capped gold nanoparticles (b).

Table 1 :
X-ray photoelectron spectroscopic data analysis for gold (Au) and iodine (I).XPS analysis shows the existence of iodine on the surfaces of gold nanoparticles.