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Department of Radiology, Sir Run Run Shaw Hospital, Zhejiang University, Hangzhou 310016, China. ο Center for Nanoparticle Research, Institute for Ba...
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Highly Sensitive Diagnosis of Small Hepatocellular Carcinoma Using pH-Responsive Iron Oxide Nanocluster Assemblies Jingxiong Lu, Jihong Sun, Fangyuan Li, Jin Wang, Jianan Liu, Dokyoon Kim, Chunhai Fan, Taeghwan Hyeon, and Daishun Ling J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b04169 • Publication Date (Web): 30 Jul 2018 Downloaded from http://pubs.acs.org on July 30, 2018

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Highly Sensitive Diagnosis of Small Hepatocellular Carcinoma Using pH-Responsive Iron Oxide Nanocluster Assemblies Jingxiong Lu,† Jihong Sun,$ Fangyuan Li,†, ‡ Jin Wang,† Jianan Liu,ο Dokyoon Kim,ο Chunhai Fan,€ Taeghwan Hyeon,ο, # and Daishun Ling†, ‡, * † Zhejiang

Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, ‡ MOE Key Laboratory of Biomedical Engineering, College of Biomedical Engineering & Instrument Science, Zhejiang University, Hangzhou 310058, China. $ Department of Radiology, Sir Run Run Shaw Hospital, Zhejiang University, Hangzhou 310016, China. ο Center for Nanoparticle Research, Institute for Basic Science (IBS), Seoul 08826, Republic of Korea # School of Chemical and Biological Engineering, Seoul National University, Seoul 08826, Republic of Korea € Division of Physical Biology and Bioimaging Center, Shanghai Synchrotron Radiation Facility, CAS Key Laboratory of Interfacial Physics and Technology, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai 201800, China Supporting Information Placeholder ABSTRACT: Iron oxide nanoparticle (IONP)-based magnetic resonance imaging (MRI) contrast agents have been widely used for the diagnosis of hepatic lesions. However, current IONP-based liver-specific MRI contrast agents rely on single-phase contrast enhancement of the normal liver, which is not sensitive enough to detect early-stage small hepatocellular carcinomas (HCCs). We herein report imotif DNA-assisted pH-responsive iron oxide nanocluster assemblies (termed RIAs), which provide an inverse contrast effect to improve the distinction between normal liver and target HCC tissues. The acidic pH of the tumor microenvironment triggers the disassembly of the RIAs, which leads to a drastic decrease in their relaxivity ratio (r2/r1), thus converting the RIAs from a T2 to T1 contrast agent. This inverse contrast enhancement of normal liver darkening and HCC brightening under T1 imaging mode was validated on an orthotopic HCC model. Our design provides a novel strategy for the exploitation of the next generation intelligent MRI contrast agents.

Iron oxide nanoparticles (IONPs) have been demonstrated to be promising magnetic resonance imaging (MRI) contrast agents.1-4 Among all available IONPs, ultra-small iron oxide nanoclusters (USIONCs) with a diameter less than 4 nm have been attracting substantial attention owing to their excellent ability for T1 MRI contrast enhancement, which results from the high portion of surface iron (Fe) ions that can alter the longitudinal and transverse relaxation times of surrounding water protons.5-7 Assisted by the surface modification by functional motifs, the IONPs have evolved into a versatile MRI platform with multiple functions.8 Moreover, the aggregation of IONPs can alter their effects on the relaxation of water protons, resulting in a drastic r2 increase.9-11 The physical mechanism underlying

this T2 enhancement was explained by the increased inhomogeneity of the magnetic field around the aggregation.12 Therefore, by manipulating the aggregation states of USIONCs, T2/T1 switching could be achieved to obtain stimuli-responsive MRI.9-11 Clinically, IONPs have been applied as liver-specific T2 contrast agents,13 but are insensitive for the diagnosis of hepatocellular carcinomas (HCC), especially for small ones less than 1 cm in size.14 Diagnosis of these small earlystage HCCs accompanied by timely curative treatments can increase the median 5-year survival rate of patients from 5% to over 50%.15, 16 The accurate detection of these small HCCs remains one of the most urgent challenges in medical diagnosis.14, 16, 17 I-motif DNAs exhibit a sensitive pH-triggered structural change that results in the transformation from singlestranded to intercalated quadruple-helical structure under acidic conditions within a dynamic pH range of 5.5–7.18, 19 This range is compatible with the typical tumor environment of pH 6–7.11, 20 Herein, we propose a highly sensitive MRI strategy to enhance the contrast between the normal liver and HCC tumors through the manipulation of the aggregation states of USIONCs via i-motif DNAs (Scheme 1 and Figure 1A). An intelligent MRI contrast agent, termed responsive iron oxide nanocluster assembly (RIA), was fabricated by linking USIONCs with i-motif DNA-derived pH-responsive linkers. We reasoned that the acidic tumor environment would induce the disassembly of RIAs into well-dispersed USIONCs, resulting in the drastic shortening of the transverse relaxation time and the initiation of MRI modular switch from T2 to T1 enhancement. Scheme 1. Schematic illustration of RIA for diagnosis of small HCC. Anchor DNA-modified USIONCs were crosslinked by pH-responsive linker DNAs to fabricate the RIA, which disassembles into dispersed USIONCs in

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acidic tumor environment. Upon intravenous injection, RIAs are routinely sequestrated by Kupffer cells in liver, whereas they disassemble into dispersed USIONCs when extravasated in the acidic HCC tumor environment. Under T1-weighted MR imaging(T1WI), the normal liver appears dark due to the presence of RIAs, while the HCC tumor becomes bright by the USIONCs.

mors. The r2/r1 ratio is a general parameter used to evaluate the T1-imaging efficiency of MRI contrast agents.5 We observed a significant drop in the r2 relaxivity with the pH change from neutral to acidic, resulting in the decrease of the r2/r1 ratio, consequently leading to the transformation of the RIAs from the T2 contrast agent in the neutral environment to the T1 contrast agent in acidic conditions (Figures 1G and S5).

USIONCs with a core diameter of ~3 nm (Figure 1B) were synthesized,5 and covered with anchor DNAs21 (see Supporting Information for details). The formation of USIONCs with 7 ± 2 homogeneous anchor DNAs modified on each nanocluster was verified by electrophoresis (Figure S1). The as-prepared USIONCs were then cross-linked into RIAs (Figure 1C and S2) by the pH-responsive linkers composed of i-motif DNAs and their partially complementary strands. The hemi-protonation of cytosine-cytosine base pairs leads to the formation of a quadruplex structure, resulting in unstable hybridization of oligonucleotides that finally dissociate from each other (Figure 1A). The RIAs exhibited a number average hydrodynamic diameter of ~120 nm in phosphate buffered saline (PBS, pH 7.4), which changed to ~20 nm in 2-(Nmorpholino)ethanesulfonic acid (MES) buffer (pH 5.5) (Figures 1C, D, and S3). Kinetic analysis and responsive pH range of the RIA disassembly process were carried out by monitoring the fluorescence intensity change of the labeled fluorophore-quencher pair as an indicator of the dissociation (Figure S4). We found that it took less than 40 s (t1/2 = 12.14 s) for the RIAs to transform into dispersed USIONCs (Figure 1E) upon the pH change from 7.4 to 5.5. The detachment rate of the USIONCs to change from the aggregation state to the equilibrium state was slightly slower (t1/2 = 18.25 s) at pH 6.5 than that at pH 5.5. This prompt disassembly response assures the immediate MRI detection of the tumor in acidic environment. The responsive pH range of the RIAs falls between pH 5.5 and 7.0, which is suitable for imaging in the acidic tumor microenvironment. The MRI properties of the RIAs in different pH conditions were evaluated on a clinical 3T MRI scanner. The MRI phantom images displayed increasing brightness as pH decreases under T1-imaging mode (Figure 1F), demonstrating the potential of sensitive detection of acidic tu-

Figure 1. Characterization and MR property evaluation of RIAs in buffers with pH 7.4, 6.5, and 5.5. (A) Schematic illustration of the working principle of the pH-responsive linker. TEM images of (B) USIONCs, (C) RIAs in PBS (pH 7.4), and (D) RIA in MES (pH 5.5). Scale bar = 50 nm. (E) Kinetic analysis of RIA disassembly upon the pH changes from 7.4 to 6.5/5.5. (F) T1WI of phantoms and (G) relaxivity data (r1 and r2) of RIAs in PBS (pH 7.4) and MES (pH 6.5 and 5.5). We examined the cellular internalization of the RIAs into normal liver cells (L02), HCC cells (LM3), and RAW 264.7 macrophages. Only RAW 264.7 cells could efficiently uptake the RIAs, while no obvious internalization of the RIAs by normal or cancerous liver cells was observed (Figures 2A and S6). Moreover, the RIAs showed no cytotoxicity to normal liver cells (Figure S7). In vitro penetration of USIONCs into tumors was further evaluated with LM3 multicellular tumor spheroids (MCTSs). Most nanoclusters were found on the surface of the MCTSs for both RIAs and pH-irresponsive iron oxide nanocluster assemblies (IRIAs), where the latter were prepared by stable dsDNA linkers (Figure S8). After disassembly, the RIAs could efficiently penetrate the MCTSs (Figures 2B, 2C, and S9), whereas no IRIA was observed inside of the MCTSs (Figure S8). The size of nanoparticles plays a critical role in the tissue penetration, with smaller nanoparticles showing marked enhancement.9, 22 Therefore, the USIONCs released from the RIAs could efficiently penetrate the LM3 MCTSs, whereas the IRIAs could only be adsorbed onto the surface of the spheroids.

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Journal of the American Chemical Society ence in signal intensity between the tumor and normal liver: (Itumor - Iliver)/Io. As shown in Figure 3D, the sensitivity of the RIAs is far superior to that of the IRIAs. Observation of the sectioned liver confirms that the size and position of the HCC tumor are consistent with the MRI result (Figure 3F). Hematoxylin and eosin staining reveals the vasculature at both edges and deep inside of the tumor (Figure S10).

Figure 3. In vivo small HCC diagnosis by RIAs. T1WI of (A) RIAs and (B) IRIAs in orthotopic HCC mice. Temporal (C) signal intensity and (D) detection sensitivity changes after RIA/IRIA injection. (E) Quantitative analysis of RIA distribution in normal liver and HCC tumors. (F) Digital image of liver tissues with HCC tumor. Prussian blue staining of (G) normal liver and (H) HCC tumor 2 h after RIA injection, 400x.

Figure 2. In vitro cellular internalization and MCTS penetration of RIAs. (A) Confocal microscopy images of L02, LM3, and RAW 264.7 cells after 2-h co-incubation with Cy5-labeled RIAs. Scale bar = 80 µm. (B) Threedimensional reconstruction of confocal microscopy images and (C) confocal microscopy images at different depths of LM3 MCTSs after 8-h co-incubation with Cy5-labeled RIAs at pH 6.5. Scale bar = 40 µm. See enlarged images for more details in Figure S9. In vivo T1 MRI of orthotopic HCC using RIAs verified the inverse contrast enhancement between the normal liver and HCC (Figure 3A). Moreover, the signal reduction in the normal liver showed a similar pattern for both RIAs and IRIAs, suggesting the absence of premature RIA disassembly before reaching the HCC (Figure 3C). Nevertheless, the tumor was brightened by the RIAs 2h after the injection while there was no signal change at the HCC site after the IRIA injection (Figure 3B), which is attributed to the prompt disassembly of the RIAs at the acidic tumor site, resulting in the T1 contrast enhancement. The sensitivity of the HCC detection was determined based on the differ-

To evaluate the mechanism of the enhanced MR sensitivity by the RIAs, the HCC mice were sacrificed, and the Fe concentrations in their liver and tumor were quantified. Compared with the blank controls, both normal liver and HCC tumor showed increased Fe concentrations after the RIA injection (Figure 3E). Although the Fe concentration increase was more prominent for the normal liver, a significant concentration increase could still be observed for HCC at 2 h post-injection, which results from the accumulation of the USIONCs in HCC that enables the T1 MRI. Prussian blue staining of the HCC liver indicated the high accumulation of the RIAs in the normal liver (Figures 3G, 3H, and S11A). Most of the RIAs in the liver tissue were distributed along the walls of the sinusoid, where Kupffer cells generally reside. Indeed, the CD68 staining (as a Kupffer cell marker) co-localized with the Prussian blue staining (Fe indicator), confirming the internalization of the RIAs into the Kupffer cells (Figure S11B). The RIAs in the HCC site presented a more dispersed form localized in the interstitial spaces of HCC tumors (Figure 3H). Transmission electron microscopy (TEM) observation confirmed the in vivo structural stability of the RIAs by locating intact RIAs in hepatic sinusoid, indicating the absence of nuclease digestion of DNAs during the circulation (Figure S12). The

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locations of the USIONCs observed under TEM are consistent with the histological analysis results in Figure 3G and 3H. These data concur with the recent finding that most hard nanomaterials are sequestrated in the liver by Kupffer cells.23 Moreover, the RIA injection did not cause any pathological change in the main organs, indicating the superior safety of the RIAs (Figure S13). In summary, small HCC diagnosis was realized successfully by assembling USIONC-based T1 contrast agents into a T2 contrast agent. The cross-linking of USIONCs through pH-responsive i-motif-based linkers confers the RIAs with the ability of disassembling back into the T1 contrast agents upon encountering the acidic tumor microenvironment. Through in vitro and in vivo evaluations, we validated that the RIAs could be transformed into T1 contrast agents at the HCC site, while remaining sequestrated in the Kupffer cells of the normal liver tissue to function as T2 contrast agents. The darkening of normal liver and the simultaneous brightening of HCC under T1-imaging mode enables highly sensitive diagnosis of small HCC. We expect that this concept will facilitate the development of nextgeneration intelligent MRI contrast agents with inverse contrast enhancement properties.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Experimental details and methods, and figures (PDF).

AUTHOR INFORMATION Corresponding Author [email protected]

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Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT This work was supported with funds from the National Key Research and Development Program of China (2016YFA0203600), the National Natural Science Foundation of China (51503180, 5161101036, 51703195), and Research Center Program of Institute for Basic Science (IBS-R006-D1) in Korea.

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