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Kyungpook National University From the SelectedWorks of Dr. Md Wasi Ahmad 2014 Bovine serum albumin (BSA) and cleaved-BSA conjugated ultrasmallGd2O3nanoparticles: Synthesis, characterization, and application to MRI contrast agents Md Wasi Ahmad Cho Rong Kim Jong Su Baeck Yongmin Chang Tae Jeong Kim, et al. Available at: http://works.bepress.com/wasiahmad/11/ Colloids and Surfaces A: Physicochem. Eng. Aspects 450 (2014) 67–75 Contents lists available at ScienceDirect Colloids and Surfaces A: Physicochemical and Engineering Aspects journal homepage: www.elsevier.com/locate/colsurfa Bovine serum albumin (BSA) and cleaved-BSA conjugated ultrasmall Gd2 O3 nanoparticles: Synthesis, characterization, and application to MRI contrast agents Md. Wasi Ahmad a , Cho Rong Kim a , Jong Su Baeck b , Yongmin Chang b,d,∗ , Tae Jeong Kim c,d , Ji Eun Bae d , Kwon Seok Chae d,e , Gang Ho Lee a,d,∗ a Department of Chemistry, College of Natural Sciences, Kyungpook National University (KNU), Taegu 702-701, South Korea Department of Molecular Medicine and Medical & Biological Engineering, School of Medicine, KNU, Taegu 702-701, South Korea c Department of Applied Chemistry, College of Engineering, KNU, Taegu 702-701, South Korea d Department of Nanoscience and Nanotechnology, KNU, Taegu 702-701, South Korea e Department of Biology Education, Teacher’s College, KNU, Taegu 702-701, South Korea b h i g h l i g h t s g r a p h i c a l a b s t r a c t • BSA and C-BSA conjugated ultrasmall • • • • Gd2 O3 nanoparticles were synthesized. BSA can bind many ultrasmall Gd2 O3 nanoparticles whereas C-BSA cannot. Large water proton relaxivities were observed. High contrast MR images in a mouse liver after intravenous injection were observed. These nanoparticles are potential MRI contrast agents. a r t i c l e i n f o Article history: Received 15 November 2013 Received in revised form 23 February 2014 Accepted 1 March 2014 Available online 12 March 2014 Keywords: BSA C-BSA Ultrasmall Gd2 O3 nanoparticle Nanoparticle carrier MRI Contrast agent a b s t r a c t Bovine serum albumin (BSA) (Mn = 66.5 kD, size = 14 × 4 × 4 nm) is an attractive biological molecule for biomedical applications because of its water-solubility and bio-compatibility. It can also bind many ultrasmall nanoparticles (NPs) as confirmed in this study. We synthesized polyethylene glycol diacid (PEGD) coated ultrasmall Gd2 O3 nanoparticles (PEGD-GNPs, the core davg = 2.0 nm), which were then conjugated to BSA and cleaved-BSA (C-BSA) (i.e. BSA-PEGD-GNPs and C-BSA-PEGD-GNPs) through amide bonding. Large relaxivities were observed in both aqueous sample solutions (r1 = 6.0 s−1 mM−1 and r2 = 28.0 s−1 mM−1 for BSA-PEGD-GNPs and r1 = 7.6 s−1 mM−1 and r2 = 22.0 s−1 mM−1 for C-BSA-PEGDGNPs). Three tesla T2 magnetic resonance imaging (MRI) in a mouse after the injection of an aqueous sample solution of BSA-PEGD-GNPs into a mouse tail vein revealed significant negative contrast enhancements. Large relaxivities and in vivo MR images prove that BSA-PEGD-GNPs and C-BSA-PEGD-GNPs are potential MRI contrast agents. © 2014 Elsevier B.V. All rights reserved. Abbreviations: BSA, bovine serum albumin; C-BSA, cleaved BSA; PEGD, polyethylene glycol diacid; NP, nanoparticle; GNP, Gd2 O3 NP; BRB, Britton–Robinson buffer; PBS, phosphate buffer solution; MRI, magnetic resonance imaging; NCT, neutron capture therapy; CT, X-ray computed tomography; DTPA, diethylenetriaminepentaacetic acid; PBS, phosphate buffer solution; NHS, N-hydroxysuccinimide; EDC, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide; GPC, gel permeation chromatograph; HVEM, high voltage electron microscope; XRD, X-ray diffraction; ICPAES, inductively coupled plasma atomic emission spectrometer; FT-IR, Fourier transform-infrared; TGA, thermo-gravimetric analyzer; DU145, human prostate cancer cell; NCTC1469, normal mouse hepatocyte cell. ∗ Corresponding authors. Tel.: +82 53 950 5340; fax: +82 53 950 6330. E-mail addresses: [email protected] (Y. Chang), [email protected] (G.H. Lee). http://dx.doi.org/10.1016/j.colsurfa.2014.03.011 0927-7757/© 2014 Elsevier B.V. All rights reserved. 68 Md.W. Ahmad et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 450 (2014) 67–75 1. Introduction 2.2. Synthesis of PEGD coated ultrasmall GNPs (PEGD-GNPs) Biological molecules have attracted considerable interest for application to nanomedicine because they are fully biocompatible and water-soluble. Bovine serum albumin (BSA) is an important carrier protein in blood plasma for several ions and molecules. It has a large dimension (14 × 4 × 4 nm) and heavy mass (Mn = ∼66.5 kD). Thus, it may also carry several ultrasmall nanoparticles (NPs) with a smaller size and mass than BSA. Biological molecules have several advantages over small molecules and polymers for biomedical applications. First, the water-solubility of surface-modified NPs generally increases with increasing mass of the ligands [1,2] and thus, biological molecules will provide enhanced water-solubility for NPs. Second, the NPs conjugated to biological molecules can remain in the blood for a longer duration than free NPs and Gd-chelates, allowing longer imaging times (so called blood-pool imaging agents) and a higher likelihood of delivering NPs to the targeted areas in a body [3–6]. This study makes use of BSA and ultrasmall Gd2 O3 NPs (GNPs) for magnetic resonance imaging (MRI). Ultrasmall GNPs have shown longitudinal (r1 ) and transverse (r2 ) water proton relaxivities larger than those of Gd-chelates because of the dense population of Gd(III) in NPs [7,8]. Therefore, BSA which could bind several ultrasmall GNPs may be useful for MRI. Here, ultrasmall GNPs can be also applied to X-ray computed tomography (CT) as CT contrast agents and neutron capture therapy (NCT) as NCT agents because Gd has a large X-ray attenuation power (∼2.5 times stronger than commercial iodine CT contrast agents) [9–12] and a very large neutron capture cross section (∼254,000 barns) [13–16]. This implies that BSA conjugated ultrasmall GNPs could be also useful for CT and NCT. Previous studies on MRI using BSA include BSA[Gd-chelates]n and BSA-GNPs (d = 20–40 nm) [3–6,10,17]. Enhanced water proton relaxivities have been observed in both systems after conjugation to BSA. In addition, the in vivo application of BSA[Gd-DTPA]n (DTPA = diethylenetriaminepentaacetic acid) showed a longer circulation time in the blood than Gd-DTPA, providing longer MR imaging times in brain tumors and blood vessels in rats [3,4]. This study examined BSA and cleaved BSA (C-BSA) (<7.0 kD) conjugated ultrasmall GNPs for MRI contrast agents. Here, ultrasmall nanoparticles (d < 3 nm) are clinically important because they can be excreted through renal system [18] and therefore, have a high potential for biomedical applications. To conjugate GNPs to BSA and C-BSA, the GNPs were surface modified with polyethyleneglycol diacid (PEGD) (Mn = 600) and then conjugated to BSA and C-BSA via amide bonding. The particle sizes, surface modifications, number of ultrasmall GNPs conjugated to BSA and C-BSA, water proton relaxivities, cellular toxicities, and in vivo MR images using a mouse were characterized. The BSA conjugated GNPs were further applied to MRI in a mouse and negative contrast enhancements in 3 T T2 MR images were clearly observed. PEGD-coated ultrasmall GNPs were synthesized using one-pot process (Scheme 1a). Two separate solutions were prepared. One is a precursor solution made from 5 mmol of GdCl3 ·xH2 O in 25 mL of triethylene glycol, and the other is a NaOH solution made from 15 mmol of NaOH in 10 mL of triethylene glycol. The precursor solution was heated to 60 ◦ C with magnetic stirring under atmospheric conditions until the precursor was dissolved completely in triethylene glycol. A NaOH solution was added to the precursor solution. The mixed solution was stirred magnetically at 180 ◦ C for 4 h. The solution temperature was reduced to 80 ◦ C and 8 mmol of PEGD was added to the solution for surface coating. An excess of PEGD was used to ensure that only one –COOH group among the two –COOH groups in PEGD could be used for conjugation to ultrasmall GNPs because the other –COOH group should be free for conjugation to BSA (or C-BSA). The solution temperature was again increased to 180 ◦ C and stirred for an additional 4 h. The solution was cooled to room temperature and transferred to a 1 L beaker containing 500 mL of triply distilled water. This was stirred magnetically for 10 min and stored for a week or so until the PEGD-coated ultrasmall GNPs settled in a beaker bottom. The supernatant was decanted and the remaining sample solution was washed again with triply distilled water. This procedure was repeated three times. 2. Experimentals 2.1. Chemicals All chemicals such as GdCl3 ·xH2 O (99.9%), NaOH (>99.9%), triethylene glycol (99%), polyethylene glycol diacid (PEGD) (Mn = 600), BSA (Mn = ∼66.5 kD), phosphate buffer solution (PBS) (pH = 7.2), HCl (>99%), N-hydroxysuccinimide (NHS) (98%), 1-ethyl-3-(3dimethylaminopropyl) carbodiimide (EDC) (>97%), boric acid (>98%), phosphoric acid (>97%), and acetic acid (98%) were purchased from Sigma-Aldrich and used as received. Triply distilled water was used for both washing the products and preparing the MRI sample solutions. 2.3. Synthesis of C-BSA To prepare C-BSA, 10 mmol of l-serine and 10 mmol of lhistidine were dissolved in 300 mL of a Britton–Robinson buffer (BRB) (40 mM phosphate, 40 mM acetate, and 40 mM borate) (pH = 5–6) at 60 ◦ C and under atmospheric conditions (Scheme 1b) [19]. Here, a pH of 5–6 of the buffer solution was achieved by adding NaOH slowly to the original buffer solution. After magnetic stirring for 30 min, 156 mg of BSA was added to the solution, and the solution was stirred magnetically for 36 h at 60 ◦ C. After the reaction was complete, the water was evaporated. This cleavage reaction was repeated 10 times to obtain enough C-BSA. The masses of CBSAs were characterized by gel permeation chromatography (GPC) and the result is summarized in Supporting Information. Two major C-BSAs with masses of 6.67 and 2.01 kD were observed from GPC analysis. The C-BSA was used without further purification because the water-soluble C-BSA could not be separated from other watersoluble reagents used in the reaction. On the other hand, the water soluble reagents were later removed after conjugation of C-BSAs to PEGD-GNPs through amide bonding. 2.4. Synthesis of BSA-PEGD-GNPs and C-BSA-PEGD-GNPs BSA-PEGD-GNPs and C-BSA-PEGD-GNPs were synthesized using an EDC/NHS coupling method (Scheme 1c) [20,21]. In this reaction amide bonds were formed between –COOH of PEGD-GNPs and –NH2 of BSA (or C-BSA). The reaction was carried out at room temperature and under atmospheric conditions. Solution pH was fixed at 6.0 by adding 1 mM HCl to the original PBS with pH of 7.2. 5 mmol of EDC and 5 mmol of NHS were added to 30 mL of PBS (pH = 6). After magnetic stirring for 30 min, the PEGD-GNPs were added to the solution and the solution was stirred magnetically for 2 h. 1.5 g of BSA (or C-BSA) was added to the solution at room temperature and stirred for an additional 2 h. The product solution was transferred to a 1 L beaker containing 500 mL of triply distilled water. The resulting solution was stirred magnetically for 10 min and stored for a week until BSA-PEGD-GNPs (or C-BSA-PEGD-GNPs) settled in the bottom of the beaker. The supernatant was decanted and the remaining sample solution was washed again with triply distilled water. This procedure was repeated three times. During this process, any water-soluble impurities such as solvent and any Md.W. Ahmad et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 450 (2014) 67–75 69 Scheme 1. Syntheses of (a) the ultrasmall GNPs and PEGD-GNPs, (b) the C-BSA, and (c) the BSA-PEGD-GNPs and C-BSA-PEGD-GNPs. other reagents used in synthesis of C-BSA, were removed from the products. Portions of the samples were evaporated to a powder form in air and the remaining portions were diluted with triply distilled water to prepare aqueous sample solutions for the MRI experiments. 2.5. General characterization The particle diameters of the ultrasmall GNPs were measured with a high voltage electron microscope (HVEM) (JEOL JEMARM1300S, 1.2 MeV acceleration voltage). A copper grid (PELCO No. 160, TED PELLA, INC.) covered with an amorphous carbon membrane was placed onto a filter paper and a sample solution diluted in triply distilled water was dropped onto the copper grid using a micropipette (Eppendorf, 2–20 L). The copper grid was dried in air for one hour to remove the solvent. The crystal structure of ultrasmall GNPs was examined using an X-ray diffraction (XRD) spectrometer (Philips, X-PERT PRO MRD) with unfiltered Cu-K␣ radiation of 1.54184 Å, scanning step of 0.033◦ , and scan range of 2 = 15–100◦ . The concentration of Gd in the sample solution was determined using an inductively coupled plasma atomic emission spectrometer (ICPAES) (Thermo Jarrell Ash Co., IRIS/AP). The surface coating of the ultrasmall GNPs with PEGD and conjugation of PEGD-GNPs to BSA (or C-BSA) were investigated using a Fourier transform-infrared (FT-IR) absorption spectrometer (Mattson Instruments, Inc., Galaxy 7020A). The powder samples were dried on a hot plate at 50 ◦ C in a hood for one week to minimize the water content. To record the FT-IR absorption spectra (400–4000 cm−1 ), pellets of the powder samples in KBr were prepared. The amount of surface coating was estimated with a thermo-gravimetric analyzer (TGA) (TA Instruments, SDT-Q 600). The TGA curves of the powder samples were recorded between room temperature and 700 ◦ C while air flowed. The amounts of PEGD, BSA, and C-BSA per GNP were estimated by recording the TGA curves. Water desorption between room temperature and ∼110 ◦ C was considered in these estimations. 70 Md.W. Ahmad et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 450 (2014) 67–75 2.6. Relaxivity and map image measurement Both the longitudinal (T1 ) and transverse (T2 ) relaxation times and longitudinal (R1 ) and transverse (R2 ) map images of the aqueous sample solutions of BSA-PEGD-GNPs and C-BSA-PEGDGNPs were measured using a 1.5 T magnetic resonance imaging (MRI) instrument (GE 1.5 T Signa Advantage, GE medical system) equipped with a Knee coil (EXTREM). Aqueous sample solutions at different Gd concentrations were prepared by diluting the original sample solutions with triply distilled water. Both map images and relaxation times were measured using these solutions. The longitudinal (r1 ) and transverse (r2 ) water proton relaxivities were estimated from the slopes in the plots of 1/T1 and 1/T2 versus the Gd concentration, respectively. The typical parameters used to measure the relaxation times and map images were as follows: the external MR field (H) = 1.5 T, the temperature (T) = 22 ◦ C, the number of acquisition (NEX) = 1, the field of view (FOV) = 16 cm, the phase FOV = 1, the matrix size = 512 × 512, the slice thickness = 5 mm, the spacing gap = 0, the pixel bandwidth = 61.0547, the repetition time (TR) = 2009 ms, and the echo time (TE) = 9 ms. 2.7. In vitro cytotoxicity measurement The in vitro cytotoxicity of the aqueous sample solutions of BSA-PEGD-GNPs and C-BSA-PEGD-GNPs was measured using both human prostate cancer (DU145) and normal mouse hepatocyte (NCTC1469) cells. A CellTiter-Glo Luminescent Cell Viability Assay (Promega, WI, USA) was used to measure the cytotoxicity. In this assay, the intracellular ATP was quantified using a luminometer (Victor 3, Perkin Elmer). The cells were seeded onto a 24-well cell culture plate and incubated for 24 h (5 × 104 cell density, 500 L cells per well, 5% CO2 , 37 ◦ C). A series of test sample solutions (0, 10, 100, and 200 M) were prepared by diluting the original sample solutions with a sterile phosphate buffer saline solution. ∼2 mL of each test solution was added to the cell culture media. The treated cell culture media were then incubated for 48 h. The cell viability of each cell was determined and normalized with respect to that of the control cell with 0.0 M Gd concentration. The measurements for all test cells were repeated twice to obtain the average cell viabilities. 2.8. In vivo 3 T T2 MR image measurement A 3 T MRI instrument (SIEMENS 3.0 T MAGNETOM Trio a Tim) was used to measure the T2 spin echo (SE) images of a mouse. The animal experiment in this study was carried out under the permission and guidance of the KNU animal committee. An ICR female mouse (ICR—Institute of Cancer Research, USA) with a weight of ∼100 g was used for the MR image measurements. The mouse was anesthetized by 1.5% isoflurane in oxygen. The measurements were made before and after injecting the sample solution into a mouse tail vein. The injection dose was ∼250 L (∼0.1 mmol Gd kg−1 ). After the measurement, the mouse was revived from anesthesia, placed into a cage, and given a free access to both food and water. During the measurement, each mouse was maintained at ∼37 ◦ C using a warm water blanket. The typical measurement parameters were as follows: the H = 3 T, the Fig. 1. HVEM images of (a) and (b) ultrasmall GNPs at two different scales, (c) BSA-PEGD-GNPs, and (d) C-BSA-PEGD-GNPs. Md.W. Ahmad et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 450 (2014) 67–75 T = 37 ◦ C, the NEX = 3–4, the FOV = 60 mm, the phase FOV = 30 mm, the matrix size = 128 × 256, the slice thickness = 1 mm, the spacing gap = 0.1 mm, the TR = 2690 ms, and the TE = 37 ms. 71 3. Results and discussion through hydrogen bonding [23–25]. The estimated cell constants are a = 6.644 Å, b = 6.841 Å, c = 6.328 Å, and ˇ = 103.976◦ for TGAtreated BSA-PEGD-GNPs and a = 6.643 Å, b = 6.839 Å, c = 6.326 Å, and ˇ = 104.001◦ for TGA-treated C-BSA-PEGD-GNPs which are consistent with the values reported in PCPDFWIN [26]. 3.1. Particle diameter (d) and crystal structure of ultrasmall GNPs 3.2. Surface modification of ultrasmall GNPs Fig. 1a–d shows HVEM images of the as-prepared ultrasmall GNPs, BSA-PEGD-GNPs, and C-BSA-PEGD-GNPs. The particle diameters of the ultrasmall GNPs ranged from 1 to 3 nm with the davg of 2.0 nm (Fig. 1a and b). The HVEM image of BSA-PEGD-GNPs indicated that many ultrasmall PEGD-GNPs were conjugated to a BSA (Fig. 1c). On the other hand, the HVEM image of C-BSA-PEGDGNPs showed that many C-BSAs were conjugated to each ultrasmall PEGD-GNP (Fig. 1d). As will be discussed later, this is consistent with the numbers of PEGD-GNPs conjugated to BSA and C-BSA estimated from TGA analyses. The XRD patterns of powder samples of both BSA-PEGD-GNPs and C-BSA-PEGD-GNPs were measured before and after TGA and are provided in Fig. 2. The XRD patterns of the as-prepared powder samples were broad, due likely to ultrasmall particle diameters [22]. On the other hand, the XRD patterns of the TGA-treated powder samples revealed sharp peaks, corresponding to monoclinic GdPO4 . All peaks after TGA analysis could be assigned to monoclinic GdPO4 and the peak positions with sufficient intensities are marked with ‘*’ in Fig. 2 and the Miller index (h k l) assignments of these peaks are provided in Supporting information. The formation of GdPO4 after TGA analysis is because the EDC/NHS coupling reaction was carried out in PBS. That is, the PO4 3− ions were likely attached to amine groups of BSA-PEGD-GNPs and C-BSA-PEGD-GNPs The surface coating of ultrasmall GNPs with PEGD followed by conjugation to BSA or C-BSA was investigated by FT-IR absorption spectroscopy. As mentioned previously, one group among the two –COOH groups in each PEGD was conjugated to an ultrasmall GNP and the other was left free for amide bonding to BSA or C-BSA. These were confirmed from the two different C=O stretching vibrations in the FT-IR absorption spectrum of the PEGD-GNPs (Fig. 3a). The free –COOH was observed at 1730 cm−1 but the –COOH bonded to GNPs, at 1620 cm−1 . The peaks at 2910 cm−1 (C–H stretch) and 1110 cm−1 (C–O stretch) also confirmed that the PEGDs were bonded to ultrasmall GNPs. The peak at 3420 cm−1 in the PEGD-GNPs was assigned to the water –OH stretch. The ∼110 cm−1 red shift of the C=O stretch after bonding to GNPs from that of the free –COOH had been observed in a range of the metal oxide NPs coated with the ligands with –COOH groups [22,27–30], supporting this result. The successful amide bond formation between PEGD-GNPs and BSA (or C-BSA) was confirmed from the disappearance of a free C=O stretch at 1730 cm−1 in both BSA-PEGD-GNPs and C-BSA-PEGD-GNPs (Fig. 3b). Instead, the N–H stretch at 3440 cm−1 (overlapped with water –OH stretch) and the N–H bend at 1530 cm−1 were observed [31–33]. The reduced intensity in N–H bend in both BSA-PEGD-GNPs and C-BSA-PEGD-GNPs was, however, observed owing to the amide bonding of amine groups of BSA and C-BSA with PEGD-GNPs and the hydrogen bonding of amine groups of BSA and C-BSA with PO4 3− ions. * * : GdPO4 (a) * * after TGA * * * * * * * * * * * * * * * * * as prepared 20 40 60 Transmittance (%) Intensity (Arb. Units) (a) PEGD-GNP 1730 3420 2910 2 * 1620 1110 3000 2000 1000 -1 Wavenumber (cm ) * : GdPO4 (b) * * * * after TGA * ** * * * * * * * * * * * as prepared 20 40 60 2 Fig. 2. XRD patterns of powder samples of (a) BSA-PEGD-GNPs and (b) C-BSA-PEGDGNPs before (i.e. as-prepared) and after TGA analysis. All peaks after TGA analysis could be assigned to monoclinic GdPO4 and only the peak positions with sufficient intensities are marked with ‘*’ (Miller index (h k l) assignments are provided in Supporting information). Transmittance (%) Intensity (Arb. Units) 4000 * (b) PEGD BSA BSA-PEGD-GNP 2910 1620 1530 C-BSA-PEGD-GNP 3440 4000 3000 2000 1000 -1 Wavenumber (cm ) Fig. 3. FT-IR absorption spectra of powder samples of (a) PEGD and PEGD-GNPs and (b) BSA, BSA-PEGD-GNPs, and C-BSA-PEGD-GNPs. 72 Md.W. Ahmad et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 450 (2014) 67–75 100 30 Water desorption = 9.8% (a) (a) 28.0% 60 20 -1 40 10 -1 0 -1 r1 = 6.0 s mM 20 0 0 0.00 100 200 300 400 500 600 700 o Temperature ( C) 100 Water desorption = 4.9% 0.25 0.50 0.75 Concentration Gd (mM) 1.00 6 (b) 80 5 (b) 47.4% -1 -1 r2 = 22.0 s mM 4 -1 ) 60 1/T (s Weight (%) -1 r2 = 28.0 s mM -1 1/T (s ) Weight (%) 80 40 20 3 -1 -1 r1 = 7.6 s mM 2 1 0 100 0 (c) 100 200 300 400 500 600 700 o Temperature ( C) Weight (%) 61.7% 0.04 0.08 0.12 0.16 Concentration Gd (mM) 0.20 Fig. 5. Plots of 1/T1 and 1/T2 of the aqueous sample solutions of (a) BSA-PEGDGNPs and (b) C-BSA-PEGD-GNPs as a function of the Gd concentration. The slopes correspond to the r1 and r2 values, respectively. and 4.4 × 1.2 × 1.2, respectively, assuming a cube root dependence of the size on the mass. Therefore, C-BSA is not large enough in mass and size to bind many ultrasmall GNPs, which is similar to the polymers. 40 20 0 0.00 Water desorption = 7.3% 80 60 0 0 100 200 300 400 500 600 700 o Temperature ( C) Fig. 4. TGA curves of powder samples of (a) PEGD-GNPs, (b) BSA-PEGD-GNPs, and (c) C-BSA-PEGD-GNPs. To determine the amounts of BSA in the BSA-PEGD-GNPs and C-BSA in the C-BSA-PEGD-GNPs, the TGA curves of PEGD-GNPs, BSA-PEGD-GNPs, and C-BSA-PEGD-GNPs were recorded (Fig. 4a–c). Water desorption between room temperature and ∼110 ◦ C was considered in these estimations. The amount of PEGD was estimated to be 28.0% from the TGA curve of PEGD-GNPs (Fig. 4a). The amounts of BSA-PEGD and C-BSA-PEGD were estimated to be 47.4 and 61.7% from the TGA curves of BSA-PEGD-GNPs and CBSA-PEGD-GNPs, respectively (Fig. 4b and c). The amounts of BSA and C-BSA were estimated to be 19.4 and 33.7% by subtracting the amount of PEGD from those of PEGD-BSA and C-BSA-PEGD, respectively. Using the davg of 2.0 nm for the ultrasmall GNPs estimated from the HVEM image and assuming that their density is the same as that (=7.407 g mL−1 ) [34] of bulk Gd2 O3 , the number of GNPs conjugated to each BSA and C-BSA were estimated to be 8.8 and 0.2, respectively, which were consistent with HVEM observations (Fig. 1b and c). Therefore, BSA is a good nanoparticle carrier, but C-BSA is not. This can be explained using the sizes and masses of BSA and C-BSA. That is, the mass of ultrasmall GNPs with the davg = 2.0 nm was estimated to be 10–20 kD by calculating the volume of the ultrasmall GNPs and using the bulk density of Gd2 O3 [34], which are smaller than those of BSA (mass = 66.5 kD and size = 14 × 4 × 4 nm). On the other hand, C-BSAs with masses of 6.67 and 2.01 kD estimated from GPC have sizes of 6.5 × 1.9 × 1.9 3.3. Suggested structures of BSA-PEGD-GNP and C-BSA-PEGD-GNP As mentioned before, the conjugation between PEGD-GNP and BSA (or C-BSA) is an amide bond between –COOH of PEGD-GNP and –NH2 of BSA (or C-BSA). The BSA consists of 607 amino acids and has amino acids with a free –NH2 [35,36] that can be used for the amide bonding to PEGD-GNP. In fact, 60 lysines with a free –NH2 are in BSA. Therefore, there are plenty of free –NH2 in BSA which can be conjugated to PEGD-GNPs through the amide bonding. As described previously, ∼9 PEGD-GNPs were estimated to be conjugated to each BSA whereas ∼0.2 PEGD-GNPs, to each C-BSA. Based on these results, structures of the BSAPEGD-GNP and C-BSA-PEGD-GNP were schematically drawn in Scheme 1c. 3.4. Relaxivities and map images Magnetic properties of gadolinium oxide nanoparticles have been well characterized [37]. They are paramagnetic but have an appreciable magnetic moment at room temperature. This is because Gd(III) has seven unpaired 4f-electrons (8 S7/2 ). Therefore, appreciable r1 and r2 values are expected from sample solutions, which were in fact observed in this study. The r1 and r2 values of BSA-PEGD-GNPs were estimated to be 6.0 s−1 mM−1 and 28.0 s−1 mM−1 , respectively, from the slopes in the plot of 1/T1 and 1/T2 , as a function of the Gd concentration (Fig. 5a). In the same way, the r1 and r2 values of C-BSA-PEGD-GNPs were estimated to be 7.6 s−1 mM−1 and 22.0 s−1 mM−1 , respectively (Fig. 5b). These values are also listed in Table 1 along with those of the other chemicals Md.W. Ahmad et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 450 (2014) 67–75 73 Table 1 Water proton relaxivity (r1 and r2 )a of various chemicals. Chemical d or davg b Nc r1 r2 Hd Te Ref. Gd-DTPA BSA-GNP BSA-PEGD-GNP C-BSA-PEGD-GNP – 20–40 2.0 2.0 – – 8.8 0.21 4.1 6.7 6.0 7.6 – 38.5 28.0 22.0 0.47 4.7 1.5 1.5 38 37 22 22 [5] [10] This work This work a b c d e Unit: s−1 mM−1 . Particle diameter or average particle diameter (nm). Number of GNPs conjugated to a BSA or C-BSA. Applied MR field (T). Sample solution temperature (◦ C). for comparison. The r1 and r2 values of BSA-PEGD-GNPs and C-BSAPEGD-GNPs are larger than those [5,6] of molecular Gd-DTPA. These increased relaxivities were attributed to the high density of Gd(III) in the NPs. These larger values generally lead to a higher sensitivity for detecting diseases in the body through contrast enhancements and can also provide the same quality MR images as those of the Gdchelates at reduced doses. The r2 values are significantly larger than that of molecular Gd-DTPA, which is why only NPs are eligible as T2 MRI contrast agents, whereas molecular agents are only suitable as T1 MRI contrast agents. The r1 and r2 values of BSA-PEGDGNPs and C-BSA-PEGD-GNPs were similar to those [10] of BSA-GNP (d = 20–40 nm) measured at a higher applied MR field. On the other hand, considering that the water proton relaxivities increase with increasing applied MR field, those of the BSA-PEGD-GNPs and CBSA-PEGD-GNPs will be larger than those of BSA-GNP at the same applied MR field. This is due likely to the particle size effect of the GNP. Both aqueous solutions of BSA-PEGD-GNPs and C-BSAPEGD-GNPs showed clear dose-dependent contrast enhancements in their R1 and R2 map images (Fig. 6a and b), suggesting that these NPs are potential candidates for MRI contrast agents, which were confirmed in a mouse experiment. 3.5. In vitro cytotoxicity The in vitro cytotoxicity of the aqueous sample solutions of BSAPEGD-GNPs and C-BSA-PEGD-GNPs were measured using DU145 and NCTC1469 cells with Gd concentrations up to 200 M (Fig. 7a and b). The results showed that C-BSA-PEGD-GNPs were slightly less toxic than BSA-PEGD-GNPs. This is probably because many CBSA like polymers encapsulated the PEG-GNPs, as shown in the HVEM image (Fig. 1c), whereas many PEGD-GNPs were conjugated to each BSA on the surface of BSA, as shown in the HVEM image (Fig. 1b). Therefore, PEGD-GNPs were better protected in CBSA-PEGD-GNPs than in BSA-PEGD-GNPs. The cell viability of both samples decreased gradually with increasing Gd concentration. The cell viability of C-BSA-PEGD-GNPs at 100 M Gd reached more than 70% for both cells, whereas that of BSA-PEGD-GNPs reached ∼60% for both cells. These levels of cellular toxicity are sufficiently low to carry out in vivo MRI experiments. 3.6. In vivo 3 T T2 MR images of a mouse Because BSA could bind many ultrasmall Gd2 O3 NPs, whereas C-BSA did not, as measured by TGA, an in vivo MRI experiment 120 (a) DU145 NCTC1469 Cell Viability (%) 100 80 60 40 20 0 0 120 (b) DU145 NCTC1469 100 Cell Viability (%) 10 100 200 Concentration Gd ( M) 80 60 40 20 0 0 Fig. 6. R1 and R2 map images of aqueous sample solutions of (a) BSA-PEGD-GNPs and (b) C-BSA-PEGD-GNPs as a function of the Gd concentration. 10 100 200 Concentration Gd ( M) Fig. 7. In vitro cytotoxicity of aqueous sample solutions of (a) BSA-PEGD-GNPs and (b) C-BSA-PEGD-GNPs using both DU145 and NCTC1469 cells. 74 Md.W. Ahmad et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 450 (2014) 67–75 (2) MR relaxivity measurements revealed that both BSA-PEGDGNPs and C-BSA-PEGD-GNPs had r1 and r2 values larger than those of molecular Gd-chelates. (3) The 3 T T2 MR images after injecting an aqueous sample solution of BSA-PEGD-GNPs into the mouse tail vein showed clear negative contrast enhancements. (4) Large relaxivities and in vivo T2 MR images prove that BSAPEGD-GNPs and C-BSA-PEGD-GNPs are potential MRI contrast agents. These results suggest that biological molecules such as BSA can be used to conjugate many surface modified ultrasmall NPs which can be applied to a variety of biomedical areas such as MRI contrast agents studied in this work. Acknowledgments Fig. 8. (a) 3 T T2 MR images of the liver of a mouse before and after injecting an aqueous sample solution of BSA-PEGD-GNPs into a mouse tail vein and (b) the plot of signal intensity in T2 MR images as a function of time after injection (0 indicates “before injection”). was further carried out using an aqueous sample solution of BSAPEGD-GNPs. Although GNPs are generally used as T1 MRI contrast agents, the T2 MR images were investigated because the r2 value was a lot larger than the r1 value, due to the appreciable magnetization of ultrasmall GNPs at room temperature [37]. 250 L (0.1 mmol Gd/kg) of an aqueous solution of BSA-PEGD-GNPs was injected into a mouse tail vein and 3 T T2 MR images of the liver were taken before and after injecting the aqueous sample solution. As shown in Fig. 8a, appreciable negative (or darker) contrast enhancements were observed in the mouse liver after the injection, which returned to almost the original contrast after 24 h due likely to the excretion of BSA-PEGD-GNPs. To more clearly see the time evolution of the contrast change in T2 MR images, the signal intensity in T2 MR images was plotted as a function of time up to 24 h in Fig. 8b. This plot clearly shows that the negative contrast enhancement maintained up to 91 min after injection but returned to almost zero above 91 min due likely to the excretion of BSAPEGD-GNPs. These results clearly indicate that the sample solution functioned as a T2 MRI contrast agent. 4. Conclusions In summary, we synthesized PEGD coated ultrasmall Gd2 O3 NPs (i.e. PEGD-GNPs) which were then conjugated to BSA and C-BSA through amide bonding (i.e. BSA-PEGD-GNPs and C-BSA-PEGDGNPs). We characterized physical and in vitro MRI properties, and cytotoxicity of BSA-PEGD-GNPs and C-BSA-PEGD-GNPs, and obtained in vivo MR images using BSA-PEGD-GNPs. (1) BSA (Mn = 66.5 kD) could bind many ultrasmall PEGD-GNPs (the core davg = 2.0 nm), showing that BSA is a good ultrasmall NP carrier. The TGA showed that ∼9 ultrasmall PEGD-GNPs could be conjugated to each BSA. However, C-BSAs (Mn < 7 kD) could not bind many ultrasmall PEGD-GNPs due to its reduced size and mass. Instead many C-BSAs were conjugated to an ultrasmall PEGD-GNP like polymers. The TGA showed that ∼5 C-BSAs were conjugated to each ultrasmall PEGD-GNP. This study was supported by the Basic Science Research Program (Grant no. 2012R1A1B3004241 to KSC, 2011-0015353 to YC, and 2013R1A1A4A03004511 to GHL) and the Basic Research Laboratory (BRL) program (Grant no. 2013R1A4A1069507) through the National Research Foundation funded by the Ministry of Education, Science, and Technology, the R&D program of MKE/KEIT (Grant no. 10040393, development and commercialization of molecular diagnostic technologies for lung cancer through clinical validation), and the KNU Research Fund (2013). The authors wish to thank the Korea Basic Science Institute for the use of their HVEM and XRD. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfa.2014. 03.011. References [1] C.J. Ackerson, P.D. Jadzinsky, R.D. 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