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Apr 21, 2016 - The in vivo MR images of PC61BA-(Gd-DO3A)/HSA-treated tumor-bearing ... Chi-Shing Tai , Carolyn J. Anderson , Peter Caravan , Ga-Lai La...
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Synergistic Effect of Human Serum Albumin and Fullerene on GdDO3A for Tumor-Targeting Imaging Ying Zhang,†,‡ Toujun Zou,†,‡ Mirong Guan,†,‡ Mingming Zhen,† Daiqin Chen,†,‡ Xiangping Guan,§ Hongbin Han,§ Chunru Wang,† and Chunying Shu*,† †

Beijing National Laboratory for Molecular Sciences, Key Laboratory of Molecular Nanostructure and Nanotechnology, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China ‡ University of Chinese Academy of Sciences, Beijing 100049, China § Department of Radiology, Peking University Third Hospital, Beijing 100083, China S Supporting Information *

ABSTRACT: A macromolecular magnetic resonance imaging (MRI) contrast agent was successfully synthesized by conjugating the gadolinium/1,4,7,10-tetraazacyclododecane-1,4,7-tetracetic acid complex (Gd-DO3A) with 6,6-phenyl-C61 butyric acid (PC61BA) and upon further modification with human serum albumin (HSA). The final product, PC61BA-(Gd-DO3A)/HSA, has a high stability and exhibits a much higher relaxivity (r1 = 89.1 mM−1 s−1 at 0.5 T, 300 K) than Gd-DO3A (r1 = 4.7 mM−1 s−1) does under the same condition, producing the synergistic positive effect of HSA and C60 on the relaxivity of Gd-DO3A. The in vivo MR images of PC61BA-(Gd-DO3A)/HSA-treated tumor-bearing mice show strong signal enhancement for the tumor area due to the enhanced permeability and retention effect. The maximum accumulation of PC61BA-(Gd-DO3A)/HSA at the tumor site was achieved at 4 h postinjection, which may guide surgery. The results from the hematology and histological observations indicate that PC61BA-(Gd-DO3A)/HSA has no obvious toxicity in vivo. These unique properties of PC61BA-(Gd-DO3A)/HSA enable them to be highly efficient for tumor-targeting MRI in vivo, possibly providing a good solution for tumor diagnosis. KEYWORDS: fullerene, macromolecular MRI contrast agent, contrast enhancement, tumor-targeting imaging, low toxicity toxicity.12−15 According to the classic Solomon−Bloembergen−Morgan theory, the crucial parameters that determine the T1 relaxivity include primarily the following three factors: the rotational correlation time (tR), the number of water molecules directly coordinated to the paramagnetic metal ions (q), and the proton residence lifetime (tm).16,17 To date, there have been enormous efforts toward the modification of ligands to achieve a longer tR, a larger q, or a shorter tm to improve the T1 relaxivity of CAs. Of these, an increase in the tR is the most feasible and commonly used method. Usually, an effective strategy for enhancing the performance of T1 CAs is either to attach the ligand with macromolecules or to incorporate multiple paramagnetic fractions into one nanoparticle.18−23 Among the diverse nanomaterials, fullerene derivatives have unique chemical, physical, and biological properties that make them an excellent unit to improve T1 CAs.24−28 For example, our group has developed (Gd-DTPA)HSA-C60 with a relaxivity of 86 mM−1 s−1 (0.5 T, 300 K);13

1. INTRODUCTION Imaging has been widely used in biomedical diagnosis due to its visual and intuitional interface.1,2 Among the diverse imaging methods, magnetic resonance imaging (MRI) is one of the most powerful techniques due to its noninvasive and nonradiative nature and the excellent visualization of anatomical details with superb spatial and temporal resolution. MRI can even reveal the molecular events related to diseases and abnormalities at the cellular and subcellular level.3−6 To improve the accuracy and specificity of MRI pathological processes, paramagnetic substances that are used as contrast agents (CAs) are commonly applied to increase the contrast between healthy and pathological tissues or to clearly emphasize the boundary of a tumor.7−10 Since the first approval of [Gd(DTPA)-(H2O)]2− by the Food and Drug Administration (FDA) in 1987, more than eight types of low molecular weight gadolinium chelates have been approved by the FDA for clinical application.3,11 However, there are still many deficiencies that limit their applications, such as low relaxivity, extracellular distribution, nonspecificity, and transient blood and tissue retention. Therefore, a high dose is required for effective diagnosis, which may result in potential © 2016 American Chemical Society

Received: December 30, 2015 Accepted: April 21, 2016 Published: April 21, 2016 11246

DOI: 10.1021/acsami.5b12848 ACS Appl. Mater. Interfaces 2016, 8, 11246−11254

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ACS Applied Materials & Interfaces

were filtered through a 0.22 μm pore size membrane before the DLS measurement. To evaluate the contrast enhancement of the obtained PC61BA-(Gd-DO3A)/HSA, the relaxivities r1 were measured at three different magnetic fields: 7 T (BioSpec70/20USR, Bruker, Germany), 3 T (Magnetom Trio, Siemens Medical Solutions, Erlangen, Germany), and 0.5 T (NMI20 Analyst instrument, Shanghai Niumag Corporation, Shanghai, China), and Gd-DO3A was measured as the control. The inversion−recovery method was introduced to measure the T1 of the samples (300 μL) for different concentrations. The relaxivities r1 were calculated via the curve fitting of 1/T1 (s−1) versus the Gd3+ ions concentration (mM). The parameters were TR = 500 ms, TE = 8 ms for 0.5 T, and 8.9 ms for 3.0 T, a slice thickness of 2.0 mm, a flip angle of 90°, a matrix of 256 × 180, and a field of view (FOV) of 200 × 150. Additionally, for the 0.5 T NMI20 analysis, a spin−echo pulse sequence was used with a pulse repetition time of D0 = 300 ms. Inductively coupled plasma mass spectrometry (ICP-MS; NexION 300X, Perkinelmer) was used to measure the concentration of the Gd3+. The serum biochemistry was measured using an automated biochemical analyzer (Nihon Kohden, Celltac E, Japan). The hematological profiles were measured using an automatic hematometer (MEK-7222K, Nihon Kohden, Japan). The histological sections were observed under an optical microscope (Olympus 71, Japan). 2.3. Transmetalation Stability Measurement. Briefly, 500 μL of PC61BA-(Gd-DO3A)/HSA (2.5 mM) in phosphate buffer (pH = 7.4, 2.5 mM) was mixed with 20 μL of ZnCl2 aqueous solution (2.5 mM), and the relaxation rate of the resulting solution was surveyed as a function of time for up to 180 h. The relative value of R1(t) at different time points t, R1(t)/R1 (t = 0), was evaluated as an indicator of the extent of transmetalation. The same measurement was taken for Gd-DO3A (2.5 mM) as a control. To examine the transchelation of Gd3+ from PC61BA-(Gd-DO3A)/HSA in vivo, 1 mL of PC61BA-(GdDO3A)/HSA (2.5 mM) that was mixed with 40 μL of ZnCl2 aqueous solution (2.5 mM) was incubated with serum. Then ICP-MS was used to measure the dissociation level of Gd3+ at different time points. 2.4. Cellular Toxicity. The 4T1 cells were cultured in DMEM that was supplemented with 10% FBS and 1% PS under a humidified atmosphere of 5% CO2. To investigate the cellular toxicity of PC61BA(DO3A-Gd)/HSA, the 4T1 cells were seeded in a 96-well culture plate with DMEM that contained 10% FBS and 1% PS in a 5% CO2 incubator at 37 °C for 24 h. Then, the aged culture medium was replaced with freshly prepared culture medium that contained PC61BA-(DO3A-Gd)/HSA solution in a series of gradient Gd3+ concentrations and incubated for another 24 h (n = 6). Subsequently, the culture medium was removed, and 100 μL of DMEM without phenol red and 10 μL of CCK-8 were added to each well. After incubation for another 1 h, the absorbance of each treated cell was measured at 450 nm using a microplate reader. 2.5. In Vitro Cellular Magnetic Resonance Imaging. The 4T1 cells (ca. 1 × 106) were cultured in a culture dish, and either GdDO3A or PC61BA-(Gd-DO3A)/HSA at different Gd3+ concentrations was added to the dish. Cells treated with PBS solution and pure water were used as the controls. After incubation for 8 h, the treated cells were washed with PBS three times, and then they were suspended in PBS buffer for the MR imaging tests. The T1-weighted cellular MR imaging were taken at 3 and 7 T, respectively, at 37 °C, using a spin− echo pulse sequence with a pulse repetition time of D0 = 500 ms. 2.6. In Vivo Tumor Magnetic Resonance Imaging. Murine breast cancer line cells (4T1 cells) were grown in DMEM medium that was supplemented with 10% FBS and 1% PS at 37 °C in 5% CO2. Female BALB/C mice (five weeks, 18−22 g) were purchased from the Beijing Xing Long Biological Technology Co. Ltd. Each mouse was subcutaneously implanted in their flanks with ca. 1 × 106 4T1 cells that were equably diffused in 50 μL of PBS. Approximately 7 d after inoculation, the mice were intravenously injected (via tail vein) with 150 μL of the complex solution at a dosage of 0.04 mmol Gd3+/kg body weight for MRI study at 7 T (TR = 200 ms, TE = 11.7 ms, FOV = 10 × 10 cm, acquisition matrix = 256 × 256, slice thickness = 1 mm), and Gd-DO3A was measured as the control.

however, the contrast enhancement and toxicity in vivo are still not available. Gao et al. synthesized C60-(Gd-DOTA)n with the relaxivity of 49.7 mM−1 s−1 at 0.5 T.29 However, the size of the fullerene nanoparticle was less than 10 nm, which was not suitable for tumor imaging through enhanced permeability and retention (EPR) effect, and the biocompatibility has not been investigated yet. Macromolecular human serum albumin (HSA) has been widely used in biomedicine, because it can carry hydrophobic particles, which can extend the blood half-life of existing drugs and reduce side effects.30−34 For example, HSA has been successfully conjugated with Gd-DTPA35 or MS-32536 to enhance the blood pool contrast, and it has been bound to a dimeric manganese porphyrin to obtain a gadolinium-free blood-pool T1 MRI contrast agent.37 The combination of HSA with fullerene has also been widely studied to investigate the biocompatibility of fullerenes.38−41 Herein, we synthesized a fullerene derivative/(Gd-DO3A) complex (PC61BA-(Gd-DO3A)) with a definite molecular structure using a simple method. Then, this complex was combined with HSA to further enhance the contrast, and to improve the biocompatibility in vivo. Notably, the short linker between C60 and DO3A and the hydrophobic−hydrophobic interaction between PC61BA-(Gd-DO3A) and HSA could limit the tumbling of Gd-DO3A and improve the resulting relaxivity. As expected, the resulting macromolecular complex, PC61BA(Gd-DO3A)/HSA, has a significantly enhanced T1 contrast ability (89 mM−1 s−1 at 0.5 T) compared to the small molecule Gd-DO3A. Additionally, the contrast ability is higher than that of the previously reported Gd-DTPA-HSA (38 mM−1 s−1 at 0.5 T) and comparable to that of Gd-DTPA-HSA-C60 (86 mM−1 s−1 at 0.5 T).13 Importantly, the obtained PC61BA-(GdDO3A)/HSA is more stable than Gd-DTPA-HSA-C60. Moreover, the aggregation size in aqueous solution is ∼70−90 nm, which is suitable for the EPR effect. All of these properties make PC61BA-(Gd-DO3A)/HSA suitable for tumor-targeting imaging.

2. MATERIALS AND METHODS 2.1. Materials. All of the chemicals and solvents were purchased from commercial sources and were analytical grade and used without further purification unless otherwise stated. The 1,4,7,10-tetraazacyclododecane, tert-butyl bromoacetate, trifluoroacetic acid, ethylenediamine, HSA, dimethyl sulfoxide (DMSO), o-(7-azabenzotriazol-1-yl)N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU), and other common solvents and reagents were purchased from SigmaAldrich Co. Ltd., China. Arsenazo III was the indicator for dissociative Gd3+ in solution, and it was purchased from Alfa-Aesar Co. Ltd., China. C60 was obtained from our laboratory (ICCAS, China). The culture media Dulbecco’s modified eagle medium (DMEM, Hyclone, United States), Cell Counting Kit-8 (CCK-8), penicillin/streptomycin (PS), and fetal bovine serum (FBS, Hyclone, USA) were all purchased from Biodee Co. Ltd., China. 2.2. Characterizations. The matrix-assisted laser desorption/ ionization time-of-flight (MALDI-TOF) mass spectra were acquired on an AXIMA Shimadzu in linear mode with dithranol as the matrix. The ultraviolet−visible (UV−vis) absorption features were observed using UV−vis spectroscopy (UV-4802H, UNIC). The Gd 3+ concentrations were measured using inductively coupled plasma atomic emission spectroscopy (ICP-AES; ICPE-9000 Shimadzu). The 1H spectra were recorded on Bruker Avance 400 spectrophotometer. The dynamic light scattering (DLS) measurements were performed on commercial LS spectrometer (ALV/DLS/SLS-5022F) at 25 °C, which was equipped with a multi-τ digital time correlator (ALV5000) and a cylindrical 22 mW UNIPHASE He−Ne laser (λ = 632.8 nm). The scattering angle was fixed at 90°. All of the samples 11247

DOI: 10.1021/acsami.5b12848 ACS Appl. Mater. Interfaces 2016, 8, 11246−11254

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ACS Applied Materials & Interfaces Scheme 1a

2.7. In Vivo Experiments. Ten mice were randomly assigned to two groups. PC61BA-(Gd-DO3A)/HSA at a dosage of 0.04 mmol Gd3+/kg body weight was intravenously injected (via tail vein) into the mice (n = 5) as the test group. The other mice (n = 5) were administered an injection of saline, and these were selected as the control group. The body weight of the mice in both groups was recorded every other day for 21 d. The mean body weights of the groups were plotted as a function of the day to show the change in weight between the control and the experimental groups. The mice were sacrificed on day 21 postinjection, and their organs were excised, washed in saline, and weighed. Organ indices (g/kg) were calculated from the ratios of the wet weights of the individual organs to the whole body weights (bw). 2.8. Hematology Examination. On the 21st day, blood samples and tissues from the mice were harvested. Blood was collected from the orbital sinus by quickly removing the eyeball from the socket with a pair of tissue forceps. Five important blood biochemistry indicators (alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), blood urea nitrogen (BUN)) and 12 useful blood routine indexes (blood platelet (PLT), neutrophil% (NE%), erythrocyte mean corpuscular volume (MCV), lymphocyte% (LY%), white blood cells (WBC), red blood cells (RBC), mean corpuscular hemoglobin (MCH), red blood cell volume distribution width (RDW), hemoglobin (HGB), hematocrit (HCT), mean corpuscular concentration (MCHC), and plateletcrit (PLT)) were measured. 2.9. Histological Examination. Upon completion of the blood collection, the mice were sacrificed. Then, the heart, liver, spleen, lung, and kidneys were removed, weighed and fixed in 4% paraformaldehyde solution, embedded in paraffin; they were then sectioned and stained with hematoxylin and eosin. The histological sections were tested for in vivo toxicity. 2.10. Organ Distribution Assay in Tumor-Bearing Mice. The mice were sacrificed at predetermined times (1 h, 4 h, 24 h, and 21 d) after injection of the PC61BA-(Gd-DO3A)/HSA through the tail vein. Organs from different animals that were exposed to the same dose were weighed and chemically digested using trace metal grade 70% nitric acid (HNO3), followed by trace metal grade 30% hydrogen peroxide (H2O2) and trace metal grade 98% sulfuric acid until only inorganic content was left. The digested samples were resuspended in trace metal grade 2% HNO3 and filtered through a 0.22 μm pore size filter membrane. The samples were examined for the Gd 3+ concentrations using ICP-MS. All animal experiments were conducted under protocols approved by Institute of Process Engineering Animal Center.

a Reagents and conditions: (a) NaHCO3/RT/48 h/diethyl ether; (b) PCBM precursor/MeONa/pyridine/reflux/24 h; (c) HCl/HOAc/ reflux/24 h; (d) DO3A/HATU/RT/24 h; (e) trifluoroacetic acid/ RT/36 h; GdCl3·6H2O/RT/12 h; (f) HSA/RT/12 h.

solution and stirred overnight at RT to produce a complexation with Gd3+. Subsequently, the product was transformed to a dialysis bag to remove the dissociative Gd3+ and DMSO. Additionally, Arsenazo III was added to the concentrated dialyzate to confirm that there was no residual dissociative Gd3+. To further improve the biocompatibility of PC61BA-(GdDO3A), HSA was introduced into the above product via a hydrophobic−hydrophobic interaction between the carbon cage and the hydrophobic cavity of HSA.39−42 The involved DMSO and excess PC61BA-(Gd-DO3A) were removed via ultrafiltration (10 000 MWCO, Millipore Corporation). The obtained brownish-red solution is highly stable in physiological media, including in water and in saline. As shown in Figure 1a and Figure S3, the UV−vis absorption of PC61BA-(Gd-DO3A)/HSA exhibits the characteristic absorption of HSA (ca. 285 nm) and PC61BA-DO3A (ca. 330 nm). The fluorescence of HSA was quenched to some extent by the attached fullerene, which is in accordance with the previous study (Figure S4, Supporting Information).43,44 These results suggest that PC61BA-(Gd-DO3A) was successfully loaded into HSA. The stoichiometry for PC61BA-(Gd-

3. RESULTS AND DISCUSSION The synthesis of PC61BA-(Gd-DO3A) is simple with a relatively high yield (Scheme 1). In brief, malonic acid modified with fullerene was first synthesized according to literature.38 Then, DO3A was attached onto the C60 exterior surface via a condensation reaction. The freshly prepared PC61BA-DO3Aboc was purified using flash column chromatography. The appearance of a m/z peak at ca. 1393 Da in the MALDI-TOFMS (Figure S1, Supporting Information) suggests the successful preparation of PC61BA-DO3A-boc, which is ca. 497 Da larger than that of PC61BA (ca. 896 Da). This was further confirmed by 1H NMR where the characteristic signals at δ = 7.26−7.98 ppm for PC61BA and at δ = 1.43 ppm for DO3A-boc appeared, as shown in Figure S2. The product was dissolved in a mixture of ice-cold trifluoroacetic acid (TFA) and CH2Cl2, then stirred at room temperature (RT) to remove the tBOC groups. In the mass spectra, the clean peak at m/z ca. 1225.9 was ascribed to PC61BA-DO3A. Additionally, the disappearance of δ = 1.43 ppm (s, 27H, tBoc) in the 1H NMR further confirmed the complete removal of tBOC. The obtained brown product was dissolved in a mixture of DMSO and GdCl3·6H2O 11248

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Figure 1. (a) UV−vis spectra of HSA, PC61BA-(Gd-DO3A), PC61BA-(Gd-DO3A)/HSA. (inset) Photographs of HSA (#1), PC61BA-(Gd-DO3A) (#2), and PC61BA-(Gd-DO3A)/HSA (#3). (b) Size distributions of PC61BA-(Gd-DO3A)/HSA in water, in saline, and in FBS. (c−e) The linear relationship between T1 relaxation rates (1/T1) and Gd3+ ion concentrations for PC61BA-(Gd-DO3A)/HSA at 0.5, 3, and 7 T, respectively. (f) T1weighted images (above) and the corresponding pseudo color images (bottom) at 7 T and 310 K.

Figure 2. (a) The comparison of r1 values of PC61BA-(Gd-DO3A)/HSA (black) and Gd-DO3A (red) at 0.5 T, 3 and 7 T, respectively. (b) Evolution of R1(t)/R1 (t = 0) as a function of time for stability evaluation of PC61BA-(Gd-DO3A)/HSA and Gd-DO3A.

T) under the same conditions in water. The T1-weighted images of PC61BA-(Gd-DO3A)/HSA exhibit a better positive contrast effect than those of Gd-DO3A under the same conditions at different magnetic fields (Figure 1f and Figure S6, Supporting Information), which is consistent with the relaxivity results. According to the previous study,29,45 the high r1 of PC61BA-(Gd-DO3A)/HSA may be attributed to the confined rotation of Gd-DO3A on C60, which leads to a relatively long rotational correlation time. Moreover, there are approximately eight PC61BA-(Gd-DO3A) molecules integrated within HSA per molecule, which can further restrict the rotation of GdDO3A via a clustering effect.36,46 The common method that is used to improve relaxivity is to increase the size of the contrast agent. Herein, we successfully used Gd-DO3A as a precursor to modify it with fullerene and HSA, which not only increased the molecular size but also restricted the small molecule’s rotation,

DO3A)/HSA was determined to be ca. 1:8 using a Braford Protein assay (Figure S5, Supporting Information) and ICPMS. The measured hydrodynamic diameters of PC61BA-(GdDO3A)/HSA are ca. 70 nm in water, ca. 90 nm in saline, and ca. 135 nm in FBS (Figure 1b), which is in agreement with previous reports.13 The protein salting out effect could lead to an increase in the size in saline due to the increase in intermolecular interactions. To evaluate the contrast enhancement of the obtained PC61BA-(Gd-DO3A)/HSA, the relaxivities r1 at three different magnetic fields were measured to be 23.3 mM−1 s−1 at 7 T, 39.2 mM−1 s−1 at 3 T, and 89.0 mM−1 s−1 at 0.5 T in water (Figure 1c−e), as well as 19.4 mM−1 s−1, 32.6 mM−1 s−1, and 78.8 mM−1 s−1, respectively, in saline, which were much larger than those of the small molecular MRI contrast agent, Gd-DO3A (3.9 mM−1 s−1 at 7 T, 4.2 mM−1 s−1 at 3 T, 4.7 mM−1 s−1 at 0.5 11249

DOI: 10.1021/acsami.5b12848 ACS Appl. Mater. Interfaces 2016, 8, 11246−11254

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decreases. This is much different from the gadofullerene MRI contrast agents, because the relaxivities of these agents significantly decrease in saline due to the disaggregation of nanoparticles.50 The salting out effect of proteins may induce a structural reconstruction of PC61BA-(Gd-DO3A)/HSA; thus, the relaxation rate decreased slightly in saline. As a result, compared with gadofullerene MRI contrast agents, such as Gd@C82(OH)n, whose relaxivities significantly decrease in saline due to the disaggregation of nanoparticles, PC61BA-(GdDO3A)/HSA exhibits better performance in a physiological environment and has brighter prospects for clinical application. The macromolecular conjugate, PC61BA-(Gd-DO3A)/HSA, has a longer resident lifetime in vivo than small molecular MRI contrast agents, which may increase the risk of potential release of free Gd3+ that is toxic from ligands. Therefore, transmetalation experiments were explored according to Muller’s method51 to investigate the stability of PC61BA-(Gd-DO3A)/ HSA. The results reveal that PC61BA-(Gd-DO3A)/HSA is more stable than that of others reported13 and than that of the small molecular MRI contrast agent Gd-DO3A for long-term. (Figure 2b) To examine the dissociation of Gd3+ from PC61BA(Gd-DO3A)/HSA in vivo, incubation with serum was also performed, and ICP-MS was used to measure the dissociation level of Gd3+ at different time points. As the result (Figure S7 Supporting Information) show, PC61BA-(Gd-DO3A)/HSA remains steady in serum, and it is suitable for in vivo imaging. As for the cellular toxicity and in vitro cellular MR imaging, the cell growth viability of the 4T1 cells that were incubated with PC61BA-(Gd-DO3A)/HSA at different Gd3+ concentrations up to 480 μM for 24 h was evaluated. As shown in Figure 3a, PC61BA-(Gd-DO3A)/HSA does not show significant cytotoxicity at concentrations ranging from 7.5 to 480 μM, indicating that the contrast agent is biocompatible in cells. T1weighted MR images of 4T1 cells that were incubated with either PC61BA-(Gd-DO3A)/HSA or Gd-DO3A and PBS were performed on a 3 and 7 T MR scanner, respectively. The results reveal that the PC61BA-(Gd-DO3A)/HSA-treated cells show more enhanced contrast in T1 imaging than Gd-DO3A treated cells both at 7 T (Figure 3b) and at 3 T (Figure S8 Supporting Information). Additionally, the signal intensity increased significantly with an increase in the Gd3+ content. According

Figure 3. (a) Viabilities of 4T1 cells after incubation with PC61BA(Gd-DO3A)/HSA (n = 6) for 24 h. (b) T1-weighted images of 4T1 cells after incubation with either PC61BA-(Gd-DO3A)/HSA or GdDO3A, PBS, and pure water for 8 h, at 7 T and 310 K.

leading to a decrease in the rotation of the entire molecule. Additionally, the relaxivity of macromolecular PC61BA-(GdDO3A)/HSA is field-strength dependent and exhibits superiority at low field (Figure 2a), which is consistent with the literature,47,48 due to the proximity of the proton Larmor frequency at the applied magnetic field strength. In contrast, the relaxivity of the small molecular MRI contrast agent (GdDO3A) is almost constant at different field strengths.49 Although the hydrodynamic diameter of PC61BA-(GdDO3A)/HSA increases slightly in saline, the relaxivity slightly

Figure 4. T1-weighted MR images of tumor-bearing BALB/C mice preinjection (a) and postinjection of PC61BA-(Gd-DO3A)/HSA (0.04 mmol Gd3+/kg bw) at 0.5 (b), 1 (c), 4 (d), 8 (e), and 24 h (f) and those of tumor-bearing BALB/C mice preinjection (a′) and postinjection of Gd-DO3A (0.04 mmol Gd3+/kg bw) at 0.5 (b′), 1 (c′), 4 (d′), 8 (e′), and 24 h (f′). Tumor tissue was highlighted in the white dotted circle. 11250

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Figure 5. (a) Changes in the bw of mice injected with either PC61BA-(Gd-DO3A)/HSA or saline (control). (b) Comparison of organ indices of the mice treated with either PC61BA-(Gd-DO3A)/HSA or saline (control). (c) H&E-stained tissue sections from mice injected with PC61BA-(GdDO3A)/HSA (above) and saline (bottom) as control. (Scale bar: 50 μm except that 100 μm for spleen). (d) Serological test results of the mice injected with PC61BA-(Gd-DO3A)/HSA and saline (control). (e) The biodistribution of PC61BA-(Gd-DO3A)/HSA in vivo.

to the previous study, the HSA−fullerene complex can be taken up by cells via the transmembrane or endocytosis, and PC61BA(Gd-DO3A)/HSA may obey the same route. The ICP results show that the cellular uptake of PC61BA-(Gd-DO3A)/HSA is ∼1.05 pg of Gd3+ per 4T1 cell, which is much more than that of Gd-DO3A (less than 0.1 picogram per 4T1 cell), suggesting its potential application for cellular trafficking and for in vivo tumor MR imaging. Then, the in vivo MR contrast enhancement of PC61BA-(GdDO3A)/HSA was further evaluated. The 4T1 tumor-bearing BALB/C mice were injected with 0.04 mmol/kg body weight Gd3+ of PC61BA-(Gd-DO3A)/HSA. For the control groups, mice were treated with the same Gd3+ amount of Gd-DO3A. The mice were narcotized using isoflurane, and in vivo MR

imaging was acquired before and after injection at 0.5, 1, 4, 8, and 24 h using the 7 T scanner. As shown in Figure 4 and Figure S9, a strong T1 contrast enhancement was observed in tumor tissues for PC61BA-(Gd-DO3A)/HSA-treated mice at 4 h postinjection, which was 350% higher than for the untreated mice. The contrast faded gradually over 24 h. The uptake of the serum proteins, especially albumin, by the tumor cells was first reported in 1948 by Mider et al.,52 which suggested that it followed an altered-tumor metabolism. The increased vascular permeability of malignant tumors along with the longer retention time of nanomaterials in blood may lead to the accumulation of the contrast agent in the interstitial space, followed by internalization in the tumor cells. Albumin-loaded PC61BA-(Gd-DO3A) probably diffuses passively into the 11251

DOI: 10.1021/acsami.5b12848 ACS Appl. Mater. Interfaces 2016, 8, 11246−11254

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scopic visual evidence, it is still difficult to quantitatively assess the PC61BA-(Gd-DO3A)/HSA-induced in vivo toxicity. The toxicity was further evaluated by analyzing the typical serum biochemical index (alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), blood urea nitrogen (BUN)) of the blood serum.59 Usually, ALT, AST, and ALP are considered to be serum indicators that quantitatively assess liver function, and BUN and LDH are often used as the indicators of kidney and heart injury, respectively. As shown in Figure 5d, these indicators for PC61BA-(Gd-DO3A)/HSA-treated mice are on a similar level to those of the control group. Several important hematology markers, such as white blood cells (WBC), red blood cells (RBC), red blood cell distribution width (RDW), erythrocyte mean corpuscular volume (MCV), etc., for the hematological assessment were also investigated.60 As shown in Table 1, none of the hematological parameters in the experimental group displayed any significant difference compared with those of the control groups. Furthermore, we investigated the biodistribution of PC61BA(Gd-DO3A)/HSA in the main organs at different time points postinjection of the materials, and the concentrations of Gd3+ in the saline group were set as the control values. After they were intravenously injected with the MRI contrast agent solution, the mice were sacrificed at 1 h, 4 h, 24 h, and 21 d postinjection. The distribution of Gd3+ in each tissue and organ are shown in Figure 5e. The results exhibited that PC61BA-(GdDO3A)/HSA nanoparticles mainly accumulated in the liver, spleen, kidney, and tumor tissues, whereas the contents in the heart, lung, and muscle were very low. At 21 d postinjection, the Gd3+ residues were undetectable in the tissues via ICP-MS, showing that most of the PC61BA-(Gd-DO3A)/HSA was excreted from the body of the mice. As mentioned above, at a dose of 0.04 mmol/kg Gd3+ of PC61BA-(Gd-DO3A)/HSA, the CA can clearly differentiate tumor tissues of BALB/c mice from healthy tissues without causing any obvious toxicity in vivo during the 21 d. According to the previous study, fullerene derivatives show good biocompatibility,61,62 and HSA from biosome is widely used in biomedical studies.31,32,63,64 Thus, as expected, the complex PC61BA-(Gd-DO3A)/HSA with good biocompatibility exhibits potential for clinical application.

Table 1. Blood Routine Examination of the Mice Injected with PC61BA-(Gd-DO3A)/HSA and Saline (Control) parameter PLTa (1 × 103/μL) NE%b (%) MCVc (μm3) LY%d (%) WBCe (1 × 103/μL) RBCf (1 × 106/μL) MCHg (pg) RDWh (%) HGBi (g/dL) HCTj (%) MCHCk (g/dL) PCTl (%)

control 810.00 19.23 58.12 68.12 7.65 9.98 16.54 15.89 18.32 60.12 30.12 0.67

± ± ± ± ± ± ± ± ± ± ± ±

33.43 2.34 3.56 6.64 1.23 0.89 0.78 0.98 0.91 3.21 0.98 0.03

PC61BA-(Gd-D03A)/HSA 773.34 17.78 60.32 65.98 8.65 9.56 17.49 14.89 17.67 56.21 32.19 0.64

± ± ± ± ± ± ± ± ± ± ± ±

43.43 1.98 3.09 3.24 1.43 0.34 1.98 1.01 0.45 2.98 0.43 0.04

a

Platelet. bNeutrophil%. cMean corpuscular volume. dLymphocyte. White blood cells. fRed blood cells. gMean corpuscular hemoglobin. h Red cell distribution width. iHemoglobin. jHematocrit. kMean corpuscular hemoglobin concentration. lPlateletcrit. e

extravascular space because of the EPR effect,53−55 and then it is internalized or metabolized by the tumor cells. Under the same conditions, no obvious difference was detected for the Gd-DO3A-treated tumor-bearing mice (Figure 4) due to the much shorter retention in tumor tissues56,57 and the rapid removal from the kidneys. To further explore the in vivo toxicity of PC61BA-(GdDO3A)/HSA, the physiological indexes of mice were also monitored during the experiment. The PC61BA-(Gd-DO3A)/ HSA solution was administered into the BALB/c mice via tail vein injection. Another group that was injected with saline was set as the control group. The fluctuation in body weight and the main organ coefficients usually are recognized as useful indicators for qualitative evaluation of toxicity in vivo. Significantly, over the recorded period during 21 d postinjection, the body weight of the mice in the experimental group normally increased from 15.5 g (0 d) to 20.5 g (21 d), which was nearly the same as that of the control group (Figure 5a). On the 21st day, the mice were all sacrificed and dissected to obtain the main organs (heart, liver, spleen, lung, and kidneys). The organ index is defined as the ratio of the wet weight of the organ (g) to the whole body weight (kg), and abnormal changes in the indices denote the reduced function of the individual organs. From Figure 5b, we observe that there is no significant difference in the organ indices between the control and the PC61BA-(Gd-DO3A)/HSA-treated groups. Histological studies are usually a reliable method to detect pathological feature changes that are due to toxicities.58 Our histopathological findings are presented in Figure 5c. Specifically, the lung sections show a similar normal alveolar geometry and a normal alveolar septum. For the liver sections, both groups show healthy hepatic architecture, hepatocytes, a portal triad, and a central vein, and no inflammatory infiltrates were observed. For the kidney sections, a normal renal cortex with normal glomerular tufts and tubules was observed. Spleen sections from both groups exhibited a normal splenic architecture with normal lymphoid follicles and sinuses, and no hydropic degeneration was observed according to the cardiac muscle tissue in the heart samples. Overall, there was no obvious inflammation, cell necrosis, or apoptosis observed, indicating that there were no obvious side effects. Although the body weight, organ coefficient, and histology provide macro-

4. CONCLUSION We developed an MRI contrast agent with Gd-DO3A, a fullerene derivative, and HSA, which proved to be a highly efficient contrast agent for tumor-targeting MRI in vivo. Importantly, the hematology and histological studies indicate that PC61BA-(Gd-DO3A)/HSA has a low toxicity in vivo. Hence, we propose a safe and effective MRI contrast agent that is hopefully extended to medical uses in future. Notably, HSA can further work as a drug delivery carrier and effectively control drug release via proteolysis. Additionally, the incorporation of fullerene in the resulting complex may further endow PC61BA-(Gd-DO3A)/HSA with photodynamic therapy properties. Therefore, this highly efficient tumor imaging hybrid may simultaneously address the optimized therapeutical time and fulfill theranostics of tumor.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.5b12848. 11252

DOI: 10.1021/acsami.5b12848 ACS Appl. Mater. Interfaces 2016, 8, 11246−11254

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Synthesis and characterizations of PC61BA-(Gd-DO3A)/ HSA: MALDI-TOF MS, 1H NMR, UV−vis spectra, fluorescence spectra, UV−vis spectra of Coomassie brilliant blue, PC61BA-(Gd-DO3A)/HSA bonding with Coomassie brilliant blue, and HSA bonding with Coomassie brilliant blue. Stability evaluation of PC61BA-(Gd-DO3A)/HSA in serum. T1-weighted images of PC61BA-(Gd-DO3A)/HSA at 0.5 and 3 T and 310 K, T1-weighted images of 4T1 cells at 3 T and 310 K, T1-weighted pseudo color MR images of tumor-bearing BALB/C mice. (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is supported by the National Natural Science Foundation of China (Nos. 51372251 and 51502301).

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