Biocompatible and Biodegradable Fe3+âMelanoidin Chelate as a

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Biocompatible and Biodegradable Fe3+–Melanoidin Chelate as a Potentially Safe Contrast Agent for Liver MRI Min-Young Lee, Dongil Choi, Moon-Sun Jang, and junghee lee Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00331 • Publication Date (Web): 01 Jun 2018 Downloaded from http://pubs.acs.org on June 1, 2018

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Bioconjugate Chemistry

Biocompatible and Biodegradable Fe3+–Melanoidin Chelate as a Potentially Safe Contrast Agent for Liver MRI

Min-Young Lee,†‡* Dongil Choi, †‡£ Moon-Sun Jang, ‡£ and Jung Hee Lee‡£*

†

Smart Healthcare Medical Device Research Center, Samsung Medical Center, 81, Irwon-ro, Gangnam-gu, Seoul, 06351, Republic of Korea

‡

Samsung Advanced Institute for Health Sciences & Technology (SAIHST), Sungkyunkwan University, 81 Irwon-ro, Gangnam-gu, Seoul, 06351, Republic of Korea

£

Department of Radiology, Samsung Medical Center, 81, Irwon-ro, Gangnam-gu, Seoul, 06351, Republic of Korea

* Corresponding authors: Tel.: +82 2 6007 5416; Fax: +82 2 6190 5396; E-mail address: [email protected] (M. Y. Lee) Tel.: +82 2 3410 6459; Fax: +82 2 3410 0084; E-mail address: [email protected] (J. Lee)

ACS Paragon Plus Environment

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Bioconjugate Chemistry 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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ABSTRACT Currently, most MRI probes available for clinical use contain gadolinium, which is a high-risk paramagnetic metal that can cause severe side effects (e.g., nephrogenic systemic fibrosis). To limit such side effects and improve diagnostic efficacy, we developed a novel biocompatible MRI contrast agent using glucose, glycine, and paramagnetic iron ion. Glucose and glycine were polymerized into melanoidin by the non-enzymatic Maillard reaction, and Fe3+ was chelated stably with the melanoidin during polymerization. The Fe3+–melanoidin chelate had biocompatibility, biodegradability, and unique contrast effects on both T1- and T2-weighted MRI, depending on the pH and oxidative environments. The administration of the Fe3+–melanoidin chelate to a mouse model of liver cancer showed highly enhanced liver-to-tumor contrasts on both T1- and T2-weighted MRI. Keywords: MRI, Contrast agent, Liver cancer, Melanoidin, Iron ion


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INTRODUCTION MRI is a noninvasive diagnostic technique that is especially useful for providing anatomical and physiological information and finding soft tissues lesions. The administration of exogenous contrast agents further improves the diagnostic efficiency of MRI in cancer and multiple sclerosis by enhancing the contrast between lesions and normal tissues.1,2 However, the low sensitivity, low specificity, and risk of side effects with the currently available contrast agents means that there is a clear need to develop new agents.3 At present, nine formulations of MRI contrast agent are approved for human use by the Food and Drug Administration (FDA), and all are based on the non-biological paramagnetic gadolinium (Gd3+) for longitudinal T1-weighted MRI.4 Due to the potential for severe toxicity with the use of free Gd3+, it must be used in a chelated form with multidentate ligands. However, these small organic ligands have fast clearance times in the body, and consequently require technically difficult procedures, such as fast image acquisition and accurate starting times, which limit specific tissue imaging.5 Despite chelation, these contrast agents can also still pose a risk of severe nephrogenic systemic fibrosis in patients with kidney dysfunction. It was recently reported that, even in healthy people, Gd3+-based contrast agents can be retained in brain or bone tissue. At these sites it is assumed to be in its free toxic state due to transmetalation with endogenous cations (e.g., Fe3+, Mg2+, Cu2+, Zn2+, or Ca2+).6 It is not known, however, whether this retained Gd3+ causes any long-term side effects. Various macromolecular organic materials (e.g., polymers7, vesicles8, and proteins9) and inorganic nanoparticles (e.g., silica10 and gold11) have been developed to solve the circulation time and toxicity problems associated with the use of Gd3+. However, their translation to clinical settings

has

often

been

frustrated

by incomplete

excretion

and

accumulation

in

reticuloendothelial organs, which can lead to the release of free Gd3+ in the long term. In recent


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research, a Gd3+-encapsulated carbon dot was developed by calcination of gadopentetic acid (GdDTPA; an FDA-approved MRI contrast agent) to reduce free Gd3+ extraction,3 but the subsequent pyrolysis of organic molecules created cytotoxic or mutagenic compounds through chemical and physical phase changes.12-15 A variety of MRI contrast agents based on ferric iron (Fe3+) have also been developed using organic materials, such as polydopamine, polyvinylpyrrolidone, and gallic acid, as chelating agents. These are effective because Fe3+, which is an essential metallic ion in the body, is considered less toxic than the non-biological paramagnetic Gd3+.3,16,17 Melanoidins are dark brown polymers commonly produced in foods by the non-enzymatic Maillard reaction of carbohydrates and amino acids through fermentation or baking.18,19 These Maillard reactions can even occur in the body,20 and we have previously synthesized melanoidin at body temperature and physiological pH to reduce the formation of cytotoxic or mutagenic compounds. Using its high light-to-heat conversion efficiency, we then developed melanoidin as a contrast agent for photoacoustic imaging and photothermal therapy.21 We also confirmed that the synthesized melanoidin had good biocompatibility, biodegradability, and renal clearance characteristics. In this research, we developed a new MRI contrast agent based on Fe3+–melanoidin chelates. Melanoidin is known to form stable complexes with metal ions because of its donor nitrogen, ketone, carboxyl, or hydroxyl groups.22 We also characterized the physical and chemical properties of Fe3+–melanoidin chelates by gel filtration chromatography, inductively coupled plasma-atomic emission spectroscopy (ICP-AES), energy dispersive X-ray spectroscopy, dynamic light scattering, atomic-force microscopy, and X-ray photoelectron spectroscopy (XPS). Finally,

cytotoxicity,

stability,

and

MR

testing

showed

excellent

biocompatibility,

biodegradability, and a unique contrast effect on T1- and T2-weighted MRI.


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RESULTS AND DISCUSSION Synthesis of Fe3+–melanoidin chelates As illustrated in Figure 1, the Fe3+–melanoidin chelates were synthesized in one step by mixing glucose, glycine, and Fe3+ in deionized water at 37°C, and adjusting to pH 7.4. To reduce the steric hindrance and to form more stable chelates, Fe3+ was chelated concurrently during the synthesis of melanoidin from glucose and glycine by the non-enzymatic Maillard reaction. As the melanoidin polymer grew, it developed the ability to chelate with Fe3+ through increases at the donor site that could satisfy the Fe3+ coordination number (6). By-products not participating in the reaction were removed through dialysis against deionized water. Gel filtration chromatography, using polyethylene glycol/polyoxyethylene as standards revealed that the molecular weights (MWs) of the Fe3+–melanoidin chelates were approximately 4900 Da after 3 days, 5700 Da after 7 days, and 7000 Da after 17 days (Figure 2a). The MWs of Fe3+–melanoidin chelates gradually increased with the reaction time. Energy dispersive X-ray spectroscopy indicated that the Fe3+–melanoidin chelates contained 39.12 atomic (atom%) C, 1.36 atom% N, 45.47 atom% O, and 14.06 atom% Fe at MW 4900 Da; 41.76 atom% C, 2.16 atom% N, 43.75 atom% O, and 12.33 atom% Fe at MW 5700 Da; and 52.14 atom% C, 3.71 atom% N, 36.91 atom% O, and 7.24 atom% Fe for MW 7000 Da (Figure 2a). As the reaction time increased, the atom% of C and N in the Fe3+–melanoidin chelate increased, while that of O and Fe decreased. Thus, the donor site appeared to be relatively reduced as the O at the edge of the formed melanoidin participated in the Maillard reaction. The elemental ratio of the Fe3+– melanoidin chelate with a MW of 7000 Da means that the reduced glucose, glycine, and Fe3+ were synthesized at a ratio of 1 : 0.5 : 1. The Fe3+–melanoidin chelate with a MW of 7000 Da can be regarded as a polymer complex composed of approximately 26 repeats of a unit containing one iron ion. The chelate with a MW of 7000 Da had 23.9 wt% of Fe3+ in ICP-AES


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analysis, which is well matched with the elemental ratio. Figure 1. Schematic of the synthesis and expected structure of Fe3+–melanoidin chelates A schematic illustration of the synthesis method and expected chemical structure of Fe3+– melanoidin chelates, as prepared using the Maillard reaction and Fe3+ chelating

Physical and chemical characterizations We investigated whether Fe3+ could be extracted from the Fe3+–melanoidin chelates by transmetalation in body fluid. The stability of Fe3+–melanoidin chelates in 100% phosphatebuffered saline (PBS, pH 7.4) and 50% fetal bovine serum (FBS 50% + PBS 50%) was monitored for 7 days, as shown in Figure S1. The Fe3+–melanoidin chelate with a MW of 4900 Da was unstable in both the 100% PBS and 50% FBS, resulting in precipitation, and the chelate with a MW of 5700 Da was stable in 100% PBS, but precipitated in 50% FBS; however, the


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chelate with a MW of 7000 Da was highly stable in both the 100% PBS and 50% FBS, even after 7 days. We thought that, during the initial polymerization, Fe3+ was chelated unstably with the edge part of melanoidin, and that as the melanoidin polymer grew, the Fe3+ was chelated stably in the intramolecular coordination sites of melanoidin. The Fe3+–melanoidin chelate with a MW of 7000 Da was therefore chosen for the further studies. We compared the chelating stabilities between two formulations: 1) the Fe3+–melanoidin chelate of Fe3+ during melanoidin polymerization, and 2) the Fe3+/melanoidin complex of Fe3+ after melanoidin synthesis. These were achieved with releasing tests for Fe3+ in either FBS or Ca2+-supplemented FBS, with the latter used because Ca2+ is known to cause transmetalation of Gd-based contrast agents.4 For the Fe3+/melanoidin complex, approximately 22.0% and 16.3% Fe3+ was released in the FBS and Ca2+-supplemented FBS, respectively; for the Fe3+–melanoidin chelate, the corresponding figures were 3.0% and 2.7% (Figure S2). Thus, Fe3+ was more stably chelated in the Fe3+–melanoidin chelate than in the Fe3+/melanoidin complex. It was also confirmed that supplemental Ca2 + did not cause transmetalation of Fe3+ that was chelated or complexed to melanoidins. As shown by atomic-force microscopy image (Figure 2b), the Fe3+– melanoidin chelate had a relatively narrow size distribution, with a vertical size of approximately 2.55 nm and a horizontal size of approximately 11.31 nm. Dynamic light scattering analysis of the Fe3+–melanoidin chelate also showed a single narrow peak with an average size of approximately 5 nm (Figure 2c). The surface of the Fe3+–melanoidin chelate had a slightly negative charge of −17.68 mV, attributable to the carboxyl groups of glycine (Figure 2d). XPS analysis of the Fe3+–melanoidin chelates was carried out to confirm the state of the Fe ion and the bonding between it and melanoidin (Figure 3a). The spectra gave binding energies of Fe 2p3/2 at 711.0 eV and Fe 2p1/2 at 724.6 eV, with satellite peaks at 718.8 eV and 729.5 eV, as well as Fe 3p at 55.9 eV, suggesting that the Fe remained in the +3 oxidation state in Fe3+–


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melanoidin chelates. The O1s spectra exhibited three peaks from melanoidin polymerization: those located at 529–530, 531.7, and 532.8 eV involved in metal-oxide, C=O, and C–O bonding, respectively. The C1s spectra exhibited three peaks at 284.5, 286.0, and 288.5 eV, corresponding to C–C, C–N/C–O, and C=O bonds, respectively. The N1s spectra exhibited one fitted peak at 400.0 eV, attributable to the C–N bond from melanoidin polymerization. The intensity-normalized XPS spectra of Fe3+–melanoidin chelates with MWs of 4900, 5700, and 7000 Da indicated that the Fe3+ state did not change with the reaction time (Figure S3). The XPS spectra of Fe3+–melanoidin chelates were broadened and shifted by melanoidin polymerization in comparison with the XPS spectra of melanoidin that was synthesized without Fe3+, glucose, and glycine (Figure S4). Fourier-transform infrared (FT-IR) spectroscopy of the Fe3+–melanoidin chelate compared to the melanoidin polymer without Fe3+ supported the coordination of Fe3+ with various groups in melanoidin. There was a peak shift of approximately 1600–1620 cm-1 (C=O, N-H coordination), a peak broadening of approximately 1240–1400 cm−1 (O-H, C-O-Metal, N-H coordination), and a peak shift and increase of approximately 980–1200 cm−1 (C-OH, N-H coordination) (Figure 3b). It was confirmed that the melanoidin polymer was synthesized from glucose and glycine, and that Fe3+ was well chelated with the melanoidin.


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Figure 2. Characterization of Fe3+–melanoidin chelates (a) Molecular weights and elemental compositions according to reaction time. (b) Atomic-force microscopy image with the vertical and horizontal size profiles. (c) Dynamic light scattering analysis. (d) Zeta potential analysis (n = 6). (a)

(b)

Elemental Composition (%)

Reaction Time (Days)

Molecular Weight (Da)

C1s

N1s

O1s

Fe2p

#1

3

4900

39.12

1.36

45.47

14.06

#2

7

5700

41.76

2.16

43.75

12.33

#3

17

7000

52.14

3.71

36.91

7.24

Name

(c)

(d)


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Figure 3. Binding energy analysis of Fe3+–melanoidin chelates (a) X-ray photoelectron spectra of Fe3+–melanoidin chelate, and (b) Fourier-transformed infrared spectra of Fe3+–melanoidin chelates compared with the melanoidin polymer. (a)

(b)


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Biocompatibility and biodegradability Before assessing the toxicity of the Fe3+–melanoidin chelate, the solubility and cytotoxicity of free Fe3+ were compared with that of free Gd3+. As shown in Figure S5, free Gd3+ did not dissolve in either PBS or FBS. This is consistent with evidence that free Gd3+ may be difficult to clear from the body when extracted from multidentate ligands.6 Moreover, as shown in the cytotoxicity test using PC12 cells in Figure S6, even if free Gd3+ failed to dissolve in the culture medium, the Gd3+ had a marked toxicity that was dependent on its concentration, producing a cell survival rate of about 25%, with morphological changes occurring at 20 mM. By contrast, although free Fe3+ was not soluble in PBS, it was soluble in FBS due to its ability to bind to serum proteins. Also, free Fe3+ did not show significant cytotoxicity, even at the high 20 mM concentration. The cytotoxicity of the Fe3+–melanoidin chelate was evaluated in comparison with GdDTPA using PC12 cells (Figure 4). During these tests, the Fe3+–melanoidin chelate did not show any significant cytotoxicity or morphological changes, even at high 40 mM concentration of Fe3+, whereas Gd-DTPA showed severe toxicity, reaching approximately 30% at a 40 mM concentration of Gd3+. For further assessment, we added Ca2+ into the media to induce transmetalation, the major cause of toxicity with conventional Gd-based contrast agents. When Gd-DTPA was incubated with Ca2+, the Gd3+ was released and precipitated in the cell medium, leading to increased toxicity, similar to that of free Gd3+. However, there was still no acute cytotoxicity and precipitation for Fe3+–melanoidin chelates, including when there was Ca2+. We carried out further cytotoxicity tests of the Fe3+–melanoidin chelate using Detroit 551 of normal human skin fibroblast. As shown in Figure S7, although the cytotoxicity was slightly lower in Detroit 551 than in PC12 cells, the results compared with Gd3+-DTPA showed similar patterns to


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those of PC12 cells. The biocompatibility of Fe3+–melanoidin chelates was thought to be due to the low cytotoxicity of both Fe3+ and melanoidin. Taken together, therefore, Fe3+–melanoidin chelates likely have superior biocompatibility to Gd3+-based agents. The biodegradability of the Fe3+–melanoidin chelate was investigated using Hydrogen peroxide (H2O2), which is presented in cells and many organs in the body. As shown in Figure S8a, the color of the Fe3+–melanoidin chelate was changed from brown to yellow in H2O2 solution, whereas the Fe3+–melanoidin chelate remained stable in pH 7.4 and pH 4.5 solutions. The UV-Vis spectra showed that the absorbance of the Fe3+–melanoidin chelate in H2O2 solution significantly reduced at 637–830 nm and increased at 500–637 nm (Figure S8b). This might be due to the decomposition of the intramolecular C=C and C=N bonds which affect long wavelength absorption.21 As shown in Figure S8c, FT-IR spectroscopy of the Fe3+–melanoidin chelate in H2O2 solution compared to the undamaged Fe3+–melanoidin chelate supported the degradation of melanoidin. There were peak shifts of approximately 1600–1640 cm-1 (C=O, N-H coordination), 1370–1390 cm-1(O-H, C-O-Metal) and 1310–1280 cm-1(N-H coordination), peak shifts and decreases of approximately 980–1200 cm−1 (C-OH, N-H coordination), and a significantly increased peak of 792 cm-1(N-H, C-H). It was confirmed that the Fe3+–melanoidin chelate was degraded in H2O2 solution.


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Figure 4. Cytotoxicity test of the Fe3+–melanoidin chelate compared with Gd-DTPA Cell viability test using PC12 cells (a) in the absence, and (b) the presence of Ca2+. (c) Its optical microscopic images. Abbreviations: Gd-DTPA, gadopentetic acid. (a)

(b)

(c)


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MR relaxation properties

In MRI, the T1 mode provides high spatial accuracy, but a low contrast-to-noise ratio, while the T2 mode exhibits high sensitivity, but generally reduces resolution and spatial specificity.23 One way to overcome these individual limitations and improve diagnostic accuracy is to use the T1 and T2 imaging modes together. To investigate the T1- and T2-weighted MRI sinal-enhancing capability of the Fe3+–melanoidin chelate, longitudinal (r1) and transverse (r2) relaxivities were measured with a 7 T clinical MRI scanner (Figure 5). The r1 and r2 relaxivities of the Fe3+–melanoidin chelate (pH 7.4) were 2.0 mM−1s−1 and 7.1 mM−1s−1, respectively. These values are comparable to those used clinically for Gd3+-based contrast agents; for example, Gd-DTPA has been reported to have an r1 of 3.5 mM−1s−1 and an r2 of 5.4 mM−1s−1 during 7 T imaging.24 The r1 and r2 can vary depending on how the paramagnetic metal ion interacts with the hydrogen ion in the body. Intramolecular transitions or intermolecular interactions of paramagnetic metal ion chelates can result in greater relaxivity changes in response to physiological microenvironments.25 The relaxivities of the Fe3+– melanoidin chelate were further investigated in pH 4.5 and H2O2 solutions to simulate lysosomal conditions and/or tumor microenvironments. The relaxivities of the Fe3+–melanoidin chelate in pH 4.5 were 2.8 mM−1s−1 (r1) and 11.0 mM−1s−1 (r2), compared with 5.5 mM−1s−1 (r1) and 64.6 mM−1s−1 (r2) in the H2O2 solution. The r1 and r2 relaxivities were higher with better T1- and T2-enhanced images compared with pH 7.4. The changes of relaxivities according to the surrounding environments can be explained mainly by the changes of the hydration number and molecular tumbling time. In the Fe3+–melanoidin chelate, the melanoidin can form pH-dependent coordination complexes with Fe3+, that is, the Fe3+–melanoidin chelate could be transformed from high number coordination at physiological pH and to low number coordination at mild acidic environment. The reduced coordination number in low pH could increase the hydration


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number in the first and second coordination spheres surrounding Fe3+, leading to enhancement of relaxivities.16 In addition, the Fe3+–melanoidin chelate can form intermolecular complex between melanoidins at low pH,22 which can enhance relaxivities by increasing the molecular tumbling time. The significant r1 and r2 enhancement in H2O2 solution could be attributed to the degradation of melanoidin. The degradation of melanoidin may lead to significant changes in the molecular structure of the Fe3+–melanoidin chelate, resulting in also highly increase of the hydration number of the first and second coordination spheres of Fe3+. The r2/r1 ratio is another important parameter for estimating whether a given contrast agent can serve as a T1 or T2 contrast agent. The r2/r1 ratios of the Fe3+–melanoidin chelate were 3.55 in the pH 7.4 solution, 3.93 in the pH 4.5 solution, and 11.75 in the H2O2 solution. Although the ratio is not roughly 1, the Fe3+–melanoidin chelate may function differently as a T1- and T2weighted MRI contrast agent depending on the tissue environment. Furthermore, T1- and T2weighted MRI of the Fe3+–melanoidin chelates revealed significant signal enhancement in a concentration-dependent manner in the pH 7.4, pH 4.5, and H2O2 solutions, showing that the Fe3+–melanoidin chelate could be an excellent candidate for use as a contrast agent of T1- and T2-weighted MRI.


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Figure 5. MRI characterization of Fe3+–melanoidin chelates Plots of (a) 1/T1 and (b) 1/T2 compared with the Fe3+ concentration of Fe3+–melanoidin chelates in pH 7.4, pH 4.5, and H2O2 solutions. (c) T1-weighted and (d) T2-weighted MRIs of Fe3+– melanoidin chelates with increasing concentrations in pH 7.4, pH 4.5, and H2O2 solutions. (a)

(b)

(c)

(d)


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In vivo MRI using animal model Gadoxetic acid is the only hepatobiliary agent currently approved by FDA and the European Medicines Agency for clinical use in liver MRI.26 However, this is another gadolinium-based contrast agent that has side effects similar to nonspecific extracellular gadolinium chelates. The superparamagnetic iron oxide nanoparticles Feridex® and Resovist® have been developed for liver imaging and approved in the United States and Europe, respectively. However, Feridex® has been withdrawn from the market and Resovist® production was discontinued due to its side effects and low imaging efficiency.27 Therefore, we evaluated the actual efficacy of the Fe3+– melanoidin chelate as a contrast agent for T1- and T2-weighted liver MRI using a mouse model of human hepatocellular carcinoma (HCC). T1- and T2-weighted liver MRIs were obtained before injection of the contrast agent and serially obtained at 0.5, 1, 2, and 24h after intravenous injection of the Fe3+–melanoidin chelate. On T1-weighted MRIs (Figure 6a), the intensities of the HCC and hepatic parenchyma were almost similar (isointense) before contrast injection, and the border of the cancer could not be determined accurately. However, when the Fe3+–melanoidin chelate was injected, the T1weighted MRIs showed a gradually brighter signal intensity (hyperintense) in the HCC until 2h, while the background hepatic parenchyma retained a darker intensity (hypointense) due to the negative contrast effect. In the serial T2-weighted MRIs, the hepatic parenchyma also retained a darker intensity (hypointensity) for up to 24 h, whereas the HCC showed relatively brighter intensity (hyperintensity) at 1h and 2 h, or little change at another time. Therefore, HCC was clearly distinguished by high contrast between the tumorous tissue and the background hepatic parenchyma at 1 h and 2 h after injection of the Fe3+–melanoidin chelate, on both T1- and T2weighted liver MRI.


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At higher concentrations of contrast agent, it has been reported that the T1 signal is saturated and starts to fall with further increases in concentration, because the T2 effects of the contrast agent start to dominate signal behavior.28 The Fe3+–melanoidin chelate appears to have a T2dominant effect in the hepatic parenchyma, but a T1-dominant effect in HCC. This might be due to the fact that the Fe3+–melanoidin chelate, with an average size of approximately 5 nm, is mainly taken up by Kupffer cells that are exclusively present in normal hepatic parenchyma, while only a small amount is taken up by liver cancer cells through the enhanced permeability and retention effect.5 In addition, T1 and T2 effects may be greatly increased by the low pH and decomposition with various reactive oxygen species after cellular uptake. These effects presumably combine to increase the contrast between a HCC and normal hepatic parenchyma on T1- and T2-weighted liver MRI. At 24 h after injection, the contrast between HCC and liver parenchyma became similar to that before the injection on both T1- and T2-weighted MRI. This may be attributed to degradation and/or clearance of the Fe3+–melanoidin chelate. To quantify the signal change, signal-to-nose ratios (SNR) were analyzed using T1-and T2 weighted MRI results as shown in Figure 6b. The difference in SNR values between normal liver tissue and tumor was greatest at 2 h after injection of the Fe3+–melanoidin chelate on both T1- and T2-weighted MRIs. The ratios of the SNRtumor/SNRliver showed that the intensity of tumor was 1.53-fold higher for T1-weighted MRI and 5.13-fold higher for T2-weighted MRI compared to that of the liver at 2 h. In addition, it was confirmed statistically significant compared with that of before injection of the Fe3+–melanoidin chelate. We conclude that the excellent biocompatibility, biodegradability, and T1 and T2 enhancement on MRI, the Fe3+–melanoidin chelate has the potential to be a MRI contrast agent for liver cancer.


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Figure 6. Representative in vivo MRI results and signal-to-noise ratio (SNR) (a) T1- and T2-weighted MRIs in a mouse model of liver cancer at different times after intravascular injection of the Fe3+–melanoidin chelate. Abbreviations: Li, liver; St, stomach. (b) SNR of tumor and liver after injection of the Fe3+–melanoidin chelate and (c) the ratio of SNRtumor/SNRliver in T1-weighted (Left) and T2-weighted (Right) MRI (n = 5). (a)

(b)

T1-Weighted

T2-Weighted


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Biodistribution The amounts of Fe3+ accumulated in main tissues of heart, lung, spleen, kidney, liver, and tumor were quantified by ICP-AES at 2 h after injection of the Fe3+–melanoidin chelate into mice model of liver cancer. The Fe3+ was accumulated in the order of the lung, spleen, kidney, liver and tumor, but was rarely accumulated in the heart (Figure S9a). The accumulation of higher amount of Fe3+ in normal liver than in tumor supported the results seen in the above in vivo liver MRI results. It was thought that, in the tumor tissues, a relatively small amount of the Fe3+–melanoidin chelate accumulates, resulting in a T1 dominant effect, wherease in normal liver tissues, a relatively large amount of Fe3+–melanoidin chelate accumulates, resulting in a T2 dominant effect. To confirm the safety of multiple/repeated imaging and exposures of the Fe3+–melanoidin chelate, we analyzed the amounts of Fe3+ remained in the major organs on the 1st and 7th days by ICP-AES after intravenously injection of the Fe3+–melanoidin chelate into normal mice. As shown in Figure S9b, Fe3+ was not accumulated in the heart at all, and Fe3+ accumulated in the lung and kidney on the 1st day was almost all released after 7 days. The Fe3+ accumulated in the liver on the 1st day remained to some extent on the 7th day, but it was confirmed that Fe3+ was definitely released as compared with that of the 1st day. The spleen still had a high concentration of Fe3+ at 7th day, probably because the Fe3+–melanoidin chelate released from the other tissues was cleared by the reticuloendothelial system (RES) of spleen. The Fe3+–melanoidin chelate accumulated in the cells can be degraded and released over time. As shown in Figure S6, even if free Fe3+ is released from the Fe3+-melanoidin chelate for prolonged periods, it may be advantageous in terms of long-term toxicity because it is significantly less toxic than free Gd3+. The molecular weight of the Fe3+–melanoidin chelate can be considered to adjust the clearance time.


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For pharmacokinetic study, the concentration of Fe3+ in plasma was measured at 0 h (preinjection), 1 h, 6 h, 24 h and 48 h after intravenously injection of the Fe3+–melanoidin chelate into normal rats. As shown in Figure S9c, Fe3+ was found to be high concentration in plasma at 2 h, but rapidly dropped in 6 h and was almost not present at 24 h. Within 6 h, some are excreted through kidney and some appear to be accumulated into tissues.

CONCLUSIONS We successfully demonstrated the efficacy of the Fe3+–melanoidin chelate based on its biocompatibility, biodegradability, and ability to function as a contrast agent of T1- and T2weighted liver MRI. The in vitro and in vivo MRI results demonstrate that the Fe3+–melanoidin chelate had good longitudinal (r1) and transverse (r2) relaxivities, with unique contrast-enhancing characteristics on T1 and T2 MRI that depended on the local environment. We also showed that the Fe3+–melanoidin chelate allowed detection of HCC with high intensity differences on T1- and T2-weighted liver MRI. The Fe3+–melanoidin chelate also benefitted from its ability to biodegrade in the cell and be efficiently excreted through hepatic clearance after systematic injection. Our observations suggest that the Fe3+–melanoidin chelate has great translation potential for use as a safe and efficient MRI probe in clinical practice. As a follow-up study, it is necessary to apply the Fe3+–melanoidin chelate to other animal disease models. Furthermore, using the light-to-heat conversion effect of melanoidin, we anticipate that the Fe3+–melanoidin chelate can be developed as a multifunctional contrast agent for use in a variety of theranostic applications.

MATERIALS AND METHODS


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Materials Glucose, glycine, iron(III) chloride hexahydrate (FeCl3·6H2O), gadolinium(III) chloride hexahydrate (GdCl3·6H2O), diethylenetriaminepentaacetic acid gadolinium(III) dihydrogen salt hydrate (Gd-DTPA), hydrogen peroxide (H2O2), calcium chloride (CaCl2), sodium hydroxide (NaOH), methyl thiazolyl diphenyl-tetrazolium bromide (MTT), and dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich (St. Louis, MO, USA). SnakeSkin dialysis tubing (3.5 K MWCO, 22 mm), RPMI 1640 medium, DMEM 11965, FBS, antibiotic–antimycotic (100X), and PBS (pH 7.4) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Hanks' Balanced Salt solution (HBSS) was purchased from Invitrogen (Carlsbad, CA, USA). Matrigel was purchased from BD Bioscience (Erembodegem, Belgium). Synthesis of Fe3+–melanoidin chelates To prepare Fe3+–melanoidin chelates, we dissolved glucose (10 g), glycine (10 g), and FeCl3·6H2O (2 g) in 100 mL of deionized water. The pH of the solution was adjusted to 7.4 using 1 M NaOH solution, before being reacted at 37°C with stirring for 3, 7, and 17 days. After reactions, the solution was purified using dialysis tubing against deionized water for 3 days and PBS (pH 7.4) for 1 day. The resulting product was stored at 4°C before use. For comparative analysis, a melanoidin polymer was prepared in a similar manner. Glucose (10 g) and glycine (10 g) were dissolved in 100 mL of deionized water, adjusted to pH 7.4 using 1 M NaOH, and reacted at 37°C by stirring for 14 days. The solution was then purified using dialysis tubing against deionized water for 3 days and freeze-dried. To prepare the Fe3+/melanoidin complex, the synthesized melanoidin (20 mg) and FeCl3·6H2O (10 mg) were dissolved in deionized water and reacted for 2 h. The solution was then purified using dialysis tubing against deionized water for 3 days and PBS (pH 7.4) for 1 day. The Fe3+ content of the Fe3+–melanoidin chelates and


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Fe3+/melanoidin complex were determined by ICP-AES. Releasing test of Fe3+ The Fe3+ concentrations of Fe3+–melanoidin chelates and Fe3+/melanoidin complexes were adjusted to 20 mM. Respectively, the solutions were dialyzed in tubing against FBS and FBS containing 5 mM of CaCl2 (adjusted to pH 7.4 by NaOH). After 3 days, the Fe3+ concentrations of the dialysis solutions were analyzed by ICP-AES. Pure FBS was used as a control. Cytotoxicity test A PC12 cell line was seeded in a 96-well plate (2 × 104 cells/well) using RPMI 1640 medium with 10% FBS and 1% antibiotic–antimycotic (100 µL/well). A Detroit 551 cell was seeded in a 96-well plate (2 × 103 cells/well) using DMEM 11965 with 10% FBS and 1% antibiotic–antimycotic (100 µL/well). After incubation for 24 h, the cells were treated with the Fe3+–melanoidin chelate at different concentrations with or without 5 mM CaCl2. For comparison, Gd-DTPA was also tested in the same manner. After incubation for 3 days, the media were washed twice using PBS and replaced with fresh media without FBS and the antibiotic–antimycotic mixture. The MTT solution (5 mg/mL) was then added (20 µL/well), and after incubation for 2 h, the media were replaced with DMSO (50 µL). Cell viability was analyzed by measuring the absorbance at 540 nm (n = 3). Mice model of liver cancer. Mice model of liver cancer were prepared by inoculation of human hepatocellular carcinoma (HCC) into BALB/c nude mice liver, as reported elsewhere.5 The mice were completely anesthetized, and a small transverse incision was made below the sternum to expose the liver. Then, a mixture of 5 µL of HBSS containing 1x106 HepG2 cells (KCLB, Korea Cell Line Bank)


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and 5 µL of Matrigel was slowly inoculated into the left upper lobe of the liver. HCC measuring

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