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Rational Design of Inflammation-Responsive Inflatable Nanogels for Ultrasound Molecular Imaging Jeongyun Heo, Chang-Keun Lim, Hyun Su Min, Kyung Eun Lee, Keunsoo Jeong, Young Hun Seo, YongDeok Lee, Ji Young Yhee, Kwangmeyung Kim, Ick Chan Kwon, Soo Young Park, and Sehoon Kim Chem. Mater., Just Accepted Manuscript • DOI: 10.1021/acs.chemmater.9b00167 • Publication Date (Web): 06 Mar 2019 Downloaded from http://pubs.acs.org on March 6, 2019

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is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Chemistry of Materials

Rational

Design

of

Inflammation-Responsive

Inflatable Nanogels for Ultrasound Molecular Imaging Jeongyun Heoa, d, ‡, Chang-Keun Lima, ‡, Hyun Su Mina, Kyung Eun Leee, Keunsoo Jeonga, Young Hun Seoa, Yong-Deok Leea, Ji Young Yheea, Kwangmeyung Kima,b, Ick Chan Kwona,b, Soo Young Parkd, Sehoon Kima,b,c,*

aCenter

for Theragnosis, Korea Institute of Science and Technology, 5 Hwarang-ro 14-gil,

Seongbuk-gu, Seoul 02792, Korea bKU-KIST

Graduate School of Converging Science and Technology, Korea University, Seoul

02841, Korea cDivision

of Bio-Medical Science & Technology, KIST School, Korea University of Science and

Technology (UST), Seoul 02792, Korea dCenter

for Supramolecular Optoelectronic Materials (CSOM), Department of Materials Science

and Engineering, Seoul National University, Seoul 08826, Republic of Korea

ACS Paragon Plus Environment

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Chemistry of Materials 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

eAdvanced

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Analysis Center, Korea Institute of Science and Technology, Hwarangno 14-gil 5,

Seongbuk-gu, Seoul 02792, Republic of Korea

ABSTRACT

Microbubbles are clinically used as an imaging agent for contrast enhanced ultrasound image. Beyond the pre-formed microbubbles, nanoscale gas-generating chemical systems that are capable of stimulus-responsive inflation to microbubbles have recently been employed as a new echogenic strategy for ultrasound molecular imaging. Here we report a peroxamide-based ultrasound contrast agent as a H2O2-responsive gas (CO2)-generating system for diagnostic ultrasound imaging of inflammatory diseases. A hydrolytic degradation-resistant peroxamide nanogel was constructed by nanoscopic crosslinking of polymeric aliphatic amines (branched polyethyleneimine) with oxalyl chloride, which intrinsically offers highly concentrated peroxamides as a reactive crosslinking point for H2O2-responsive CO2-generation by the peroxalate chemiluminescence (POCL) reaction that is intrinsically catalyzed by the polyaminederived intraparticle basic environment. It was experimentally revealed that the interior of the peroxamide-concentrated nanogel colloid serves as an optimal nanoscale catalytic reactor for the H2O2-responsive gas generation as well as a gas reservoir capable of nano-to-micro inflation. We demonstrate that the peroxamide-concentrated nanogels are indeed capable of enhancing the ultrasound contrast in response to H2O2, which allows us to perform diagnostic ultrasound imaging of H2O2-overproducing inflammatory diseases in mouse models. Along with the biocompatibility of the peroxamide nanogels revealed by the animal toxicity study, our design


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strategy for the inflatable nanoparticles would contribute to the advancement of activatable contrast agents for ultrasound molecular imaging.

INTRODUCTION Ultrasound (US) imaging is the most commonly used clinical modality that is safe, inexpensive and highly sensitive with real-time speeds, millimeter resolution, and long imaging depth.1, 2 Among its limitations is inherently poor image contrast that stems from similar acoustic impedances between normal and abnormal soft tissues. One way to overcome this drawback is to use ultrasound contrast agents (UCAs) that are highly echogenic with a large mismatch of acoustic impedance with tissues. Typical UCAs are pre-formed microbubbles (> 1 =

in

diameter) for angiography that are filled with gas molecules as a highly echogenic contrast medium. For phenotype-targeted US molecular imaging, current methods rely on surface engineering of the pre-formed microbubbles by ligand conjugation and their selective accumulation at pathological tissues through binding to disease-specific cell membrane receptors.3-6. However, such a targeting mechanism using cell surface binding of pre-formed microbubbles is not available for targeting cell-secreted or intracellular biomarkers. To complement this, stimulus-responsive gas-generating chemical systems have recently been employed as an alternative echogenic strategy for activatable US molecular imaging. Activatable UCAs are initially silent with no echogenicity but become echogenic in pathological tissues where gas molecules (CO2, O2, or N2, etc.) form and coalesce into echogenic microbubbles by reaction with disease-specific phenotypes such as abnormal pH or overproduced metabolites.7-12 Therefore, selective post-formation of microbubbles by activatable UCAs allows for molecular


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imaging of phenotypes that cannot be imaged by ligand-receptor binding on the cell surface. Moreover, this strategy holds potential for enhancing the image contrast by minimizing background signals, because untargeted/unreacted UCAs in the surrounding normal tissues are kept non-echogenic. Being a molecular hallmark of numerous inflammatory diseases, hydrogen peroxide (H2O2) is recognized as a valuable cell-secreted diagnostic marker that is aberrantly overproduced in the progress of inflammation.13,14 Although various signals (ex. fluorescence and chemiluminescence) have been employed for detecting H2O2,15-18 a more practical modality is in high demand for diagnostic imaging in the clinical setting. In this regard, activatable US imaging of H2O2 has been pioneered with catalase-like inorganic nanoparticles that generate gas microbubbles by catalyzing breakdown of H2O2 into O2 molecules.10,11,19 More recently, peroxalate polymer nanoparticles have been reported, which proved the concept that the H2O2specific reaction of peroxalates is applicable to generate echogenic CO2 bubbles. 20, 21 Molecular US imaging of inflammation, if established in the clinic, would lead to great advancement in medical imaging; hence, there are growing needs of extensive researches on a wide spectrum of H2O2-responsive chemical systems to optimize the echogenic microbubble formation. As exemplified above, peroxalate derivatives are a class of reactive molecules that are known to produce CO2 molecules as a byproduct during the well-known peroxalate chemiluminescence (POCL) reaction.22 They react selectively with H2O2 to produce chemiluminescence (CL) and CO2 molecules as a result of decomposition of the high-energy intermediate, 1,2-dioxetanedione (Figure 1a). Peroxalate esters are a representative family with high reactivity and have successfully been demonstrated to be useful for in vivo CL imaging of inflammatory H2O2 in a form of water-dispersed nanoparticles.16-18 Therefore, they hold potential


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as a promising chemical system of H2O2-responsive gas (CO2) generation for US imaging; however, most of peroxalate esters are chemically unstable against hydrolysis, which critically limits their biomedical utility in aqueous media. Moreover, compared to the intrinsically sensitive CL modality, the sensitivity of US signaling would be determined by its complex contrast mechanism that does not only rely on gas generation but also on subsequent gas coalescence into echogenic bubbles. Consequently, though the CL emission and gas generation by the peroxalate reaction share the same mechanism, a more elaborate and rational design strategy is demanded to be drawn for advancing peroxalate-based UCAs and US molecular imaging of inflammation thereby. In this study, we explored chemical aspects of the water-dispersed peroxalate-based UCA, the interior of which serves as a nanoscale reactor for the H2O2-peroxalate reaction as well as a gas reservoir capable of nano-to-micro inflation. To resolve the hydrolytic instability issue of peroxalates in water, we employed an aliphatic amide derivative (peroxamide) as a chemically stable alternative. Peroxamides are known to be chemically stable against hydrolysis, but at the same time, they are poorly reactive toward H2O2 particularly in the case of aliphatic peroxamides,23 which necessitates the incorporation of a catalytic mechanism to attain efficient POCL reaction in the poorly reactive medium. Taking these issues into consideration, we devised a rational design of peroxamide-concentrated nanogels as an efficient UCA for H2O2 imaging. In our design, a polymeric aliphatic amine was crosslinked via peroxamidation to form a densely concentrated H2O2-reactive CO2-generating source (Figure 1b), where uncrosslinked remnant amines serve as an in situ basic catalyst for POCL reaction. The crosslinked aliphatic peroxamide was further formulated into water-dispersed nanogel colloids as a nanoscale UCA (USNG; Figure 1c). We studied the effects of USNG composition on the activatable contrast-


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enhancing performance for US imaging, which is determined by the efficiencies of the H2O2responsive generation/capturing of CO2 gas molecules in the peroxamide nanoreactor and thereby its inflation into a stable echogenic microbubble. As a proof of concept, we applied an optimized formulation of perfluorocarbon-containing USNG to diagnostic imaging of inflammatory diseases in mouse models (hepatitis and cancer), which validates the value of our design concept for the advancement of US molecular imaging.

RESULTS AND DISCUSSION The network structure of crosslinked aliphatic peroxamide was formed by amidation of branched PEI (bPEI) with oxalyl chloride (Figure 1b). We employed highly branched low molecular weight bPEI (Mw = 0.6 k) as a polymeric aliphatic amine because of (1) its extremely high density of reactive amine groups for crosslinking, which facilitates the preparation of a dense network structure where reactive peroxamides are concentrated and stably incorporated as a crosslinking point, (2) possibility of imposing a basic intraparticle environment by unamidated remnant amines of bPEI, which would promote the peroxamidation reaction as well as the POCL reaction of the crosslinked peroxamides by in situ basic catalysis without additional catalysts, (3) fairly reduced toxicity compared to the higher molecular weight derivatives,24 and (4) high CO2 affinity of amines that is advantageous for stable gas capture and microbubble inflation.25,26 bPEI contains primary (pKa = 4.5), secondary (pKa = 6.7), and tertiary (pKa = 11.6) amines in a ratio of 1:2:1, where primary and secondary amines are available for amidation with oxalyl chloride. The feasibility of gel formation by peroxamidation was simply confirmed by bulk reaction in an anhydrous organic medium. When bPEI and oxyalyl chloride were mixed in methylene chloride,


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an immobile bulk gel was instantaneously formed at room temperature (Figure 1d), indicating that crosslinking of bPEI with oxalyl chloride is highly efficient under in situ basic catalysis. To prepare nanoscale bPEI-peroxamide gels, the gelation reaction was conducted in a confined nanoscopic space by using anhydrous reverse micelles of Tween 80 to form neat bPEI droplets in cyclohexane (Figure S1). After suspension breaking with ethanol, the obtained nanogel was purified by repeated centrifugation and ethanol washing for complete removal of Tween 80 and extra impurities. The oxamide bond formation in the bulk and nanoscale gels was verified by IR spectroscopy (Figure 1e). Compared to the spectrum of bPEI, both of the gel products showed three new vibronic bands at (i) 1509 (N-H bending of oxamide), (ii) 1666 (C=O stretching of oxamide) and (iii) 1734 cm-1 (C=O stretching of oxalic acid derived from the unreacted acyl chloride side of half-amidated products)27. Nanogel colloids as a H2O2-activatable nanoscale UCA (USNG) were further formulated by redispersing the dried bPEI-peroxamide nanogels in water with Pluronic F-68 as a colloid stabilizer and perfluorohexane (PFH) as an intraparticle medium with a high CO2-capturing capacity28 (Figure 1c). The obtained USNG showed good dispersability in water, as analyzed with colloidal morphology by cryotransmission electron microscopy (cryo-TEM; 110 ± 23 nm in diameter, Figure 1f) and hydrodynamic size distribution (112 ± 72 nm) by dynamic light scattering (DLS; Figure S2), and maintained stably in water over time (Figure S3). It was confirmed that the zeta potential of naked bPEI-peroxamide nanogel is highly positive (~35 mV) owing to the exposure of PEI on the surface, whereas that of USNG is significantly attenuated toward neutral (~8 mV) by further formulation with Pluronic and PFH (Figure S4). The reduced positive surface charge of the Pluronic-formulated USNG would help prevent non-specific recognition by organs of the reticuloendothelial system (RES) such as liver and spleen, being useful for in vivo applications.29


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Since decomposition of 1,2-dioxetanedione to CO2 coincides with the CL emission in the POCL mechanism, the capability of bPEI-peroxamide gels for H2O2-responsive CO2 generation was evaluated indirectly by monitoring the CL emission of USNG (Figure 1a). To render USNG chemiluminescent, bPEI-peroxamide nanogels were labeled with a fluorescent dye (Cy5.5) as a CL emitter at the remnant amines of bPEI and further formulated into USNG with optimized composition of F-68 and PFH (vide infra). It was observed that the addition of excess H2O2 (100 mM, 0.2 mL) to the colloidal USNG suspension (1 mL) generates the CL emission without photoexcitation, which spectrally matches with the photoluminescence (PL) of Cy5.5 attached to the nanogel (Figure S5). Firstly, we measured the CL intensity at different pHs to examine the pH effect on in situ basic catalysis of the nanogel (Figure 2a). It is known that general base catalysis can promote the POCL reaction by transforming H2O2 into a more reactive deprotonated form.30, 31 Although USNG is composed of poorly reactive aliphatic peroxamides, it showed clear CL emission even at a near-neutral physiological pH of 7.4 which is not basic enough for catalysis. This suggests that the intraparticle environment is catalytically active by itself due to the intrinsic basicity of the nanogel imposed from the remnant amines of bPEI. Indeed, the CL emission dimmed significantly at an acidic pH of 4.5 where all kinds of bPEI amines would be protonated to lose basicity. These pH responses imply that the POCL reaction of aliphatic peroxamides are indeed catalyzed by the intrinsic basicity of the nanogel, and also that such intrinsic basicity is susceptible to the acidic environment to lose the catalytic activity. Notably, the weak CL intensity at pH 4.5 could be recovered by re-neutralization to pH 7.4, likely due to the regeneration of basic amines. Considering that the PL intensities of Cy5.5 at both the pHs were measured almost the same (Figure 2b), it is suggested that the pH response of Cy5.5 itself has no contribution to the observed pH-dependent CL alteration. Therefore, all these


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results unambiguously conclude that the nanogel of poorly reactive aliphatic peroxamides is selfcatalyzed by the intrinsic basicity to facilitate the intraparticle POCL reaction. To corroborate the catalytic effect of bPEI in the nanogel, we further examined the reaction kinetics by using a model system in solution. As shown in Figure 2c, dipropyloxamide (DPO) was employed as a low-molecular-weight model compound for the aliphatic peroxamide backbone of the nanogel. The POCL profiles of DPO in solution with or without bPEI were analyzed by following the pseudo-first-order reaction kinetics (Figure S6) and compared with that of a highly reactive peroxalate ester, bis[3,4,6-trichloro-2-(pentyloxycarbonyl)phenly] oxalate (CPPO). As indicated with the rise kinetic rates (kr) in Figure 2d, DPO alone showed a very slow POCL reaction with H2O2, representing the low POCL reactivity of aliphatic peroxamides as expected. In the presence of basic bPEI, however, the rate constant (kr) was remarkably increased with increasing the amount of bPEI. Importantly, when DPO was reacted in the presence of a high concentration of bPEI (20 mg) that is close to the intraparticle amount of the remnant bPEI amines in the peroxamide nanogel, it was shown that the POCL reaction of an aliphatic peroxamide could be even faster than that of reactive peroxalate CPPO. These results support the occurrence of in situ basic catalysis by bPEI in USNG to strongly enhance the POCL reactivity for H2O2-responsive CO2 generation. A main purpose of employing the peroxamide structure is to replace easily hydrolyzable peroxalate esters and attain the long-term chemical stability of USNG against hydrolytic degradation to secure the practical utility for US imaging. To evaluate the storage stability, we again monitored the POCL behavior of USNG in comparison to water-dispersed nanoparticles of peroxalate ester (CPPO). Each sample was stored at 4 °C, and aliquots were temporally subjected to a room-temperature reaction with H2O2 at designated time points up to 4 d, where the


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measured CL intensity reflects the POCL reactivity maintained during the storage (Figure 2e). As anticipated, the CPPO nanoparticles showed a gradual drop of the CL intensity from the beginning and turned no more chemiluminescent 6 h after exposure to water for colloidal dispersion, indicating a rapid decline of the POCL reactivity by hydrolysis of peroxalate ester. In sharp contrast, the CL intensity of USNG was persistent for at least 4 d without any notable sign of decline, affirming that the peroxamide structure is chemically robust enough to survive the hydrolysis in physiological media. We then microscopically examined the nano-to-microscale inflation behavior of USNG to evaluate the capability to form microbubbles as an echogenic contrast medium. Figure 3a shows the optical micrograms of USNG dispersed in PBS (pH 7.4), where only microscale features are detectable on the given optical resolution. Without H2O2 added, nothing was observed from the USNG dispersion in PBS (pH 7.4), indicating that water-dispersed USNG alone maintains its nanoscale structure without undergoing inflation. After adding H2O2, however, a number of optically observable microbubbles were formed within 30 s of POCL reaction, and some of them inflated as large as 20 m with time. The blank control formulation, which is composed of bPEI, F-68 and PFH but does not include the bPEI-peroxamide structure, was shown to form no noticeable micron-size bubbles even after the addition of H2O2 (Figure S7), suggesting that the microbubble generation from USNG is attributed to the CO2 production through the POCL reaction of peroxamide. This observation could be corroborated by cryoscanning electron microscopy (cryo-SEM) using a freeze-fracturing technique. As shown in Figure 3b, no particulate objects were seen in the cryo-SEM image prior to POCL reaction, whereas spherical microbubble structures were clearly observed from the H2O2-reacted USNG sample. To chemically and qualitatively confirm the production of CO2 gas during the reaction,


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mass spectrometry was conducted for dried bPEI-peroxamide under carrier gas flow (He, 30 cm3/min) at room temperature. Due to the limited detection sensitivity of the mass spectrometry system, we added an excess amount of H2O2 as much as 2~3 drops (30 w% in water, ~ 0.8 M) to ensure an enough quantity of gas to be generated. Upon monitoring at the mass peak of CO2 at 44 Da (Figure S8), the partial pressure of CO2 became 3.2-fold higher when the bPEIperoxamide was mixed with H2O2 (Figure 3c), directly evidencing that the gas molecule responsible for microbubble inflation is CO2, i.e., the gaseous side product of POCL reaction. Figure 3d shows the in vitro US phantom images of USNG before and after adding H2O2. In the absence of H2O2, acoustic contrast of the USNG suspension was measured very weak, whereas it was enhanced by about 5 times immediately after adding H2O2 (Figure 3e); the enhanced punctate signals were seen floating with gradually increasing buoyancy with time, corroborating that the contrast enhancement is attributed to the H2O2-responsive inflating bubbles. It turned out that the H2O2-induced contrast-enhancing performance of USNG is susceptible to its composition. Among various compositions examined, the best acoustic reflectivity in the presence of H2O2 was achieved from the one composed of 10 mg bPEIperoxamide, 20 mg F-68 and 1

L PFH (Figure S9), which was employed as an optimized

USNG formulation for all other experiments in this study. Having confirmed the H2O2-responsive microbubble generation and concomitant US contrast enhancement, we applied the above-optimized formulation of USNG to diagnostic US imaging of inflammatory diseases in murine models. In clinical practice, US imaging is widely employed as a diagnostic modality for liver diseases and cancer, where the alteration of acoustic impedance in the pathological tissues is noticeable in the sonographic examination.32 In addition, it is now generally accepted that ROS are generated as a result of inflammatory process in the


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pathological conditions of liver inflammation33 and tumor.34 In this regard, we employed murine models of acute liver injury and squamous cell carcinoma (SCC) to evaluate the diagnostic potential of USNG. For these local diseases, we applied USNG via local administration that in general offers clinical benefits with potential to significantly increase the efficacy while minimizing systemic toxicity by delivering active ingredients directly to the site of action.35 Firstly, acute liver injury was induced by intravenous injection of an endotoxin, lipopolysaccharide (LPS), to BALB/c mice primed with Propionibacterium acnes,36 and confirmed by histologic analysis and blood test (Figure S10). For comparative monitoring of US responses, USNG was carefully injected into the liver tissues of normal and injured mice under US imaging guidance. Immediately after administration, the injured liver tissue exhibited clearly enhanced and long-lasting US contrasts that were seen dominantly in the blood stream along liver vessels, whereas no contrast change was noticed in the normal mice (Figure 4a, Movie S1S4). Such distinct responses are well correlated with the histologic analysis (Figure S10b); i.e., compared to the normal control mice, the injured liver tissues of the LPS-treated group showed typical histologic features of inflammation such as widespread focal hepatocellular necrosis and a large number of infiltrated mononuclear immunocytes. In particular, lymphocytes or plasma cells were shown to accumulate around the blood vessels. It is thus reasonable to deduce that the nano-sized USNG is subjected to drainage from the liver tissue into well-developed networks of capillaries and small veins in the liver structure; during the drainage, it would react with a high level of inflammatory ROS from the immunocytes and damaged cells and eventually form microbubbles in the vessels to enhance the US contrast. Hence the signal enhancement reflects the oxidative stress in the inflamed liver tissue. It is noted that even before USNG injection, the injured liver parenchyma showed notably enhanced contrasts in the sonography by itself


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compared to normal liver (Figure S10b), which is attributed to histological changes by inflammation.37,

38

In spite of such a pre-enhanced background, H2O2-responsive USNG

produced 3 times enhanced sonographic contrasts in the inflamed liver vessels, providing a more evident diagnostic distinction between injured and normal liver tissues. Next, we evaluated the potential of USNG for cancer diagnosis. In the clinic, tumor size is an important parameter for finding the best treatment option. Although US imaging is a convenient tool for tumor size estimation, it is known that tumors smaller than ~2 cm are hard to distinguish by US contrast.39 In this regard, the inflammation-mediated contrast enhancement by USNG would allow for more precise diagnostic imaging of tumors. To prove this, USNG was intratumorally administered to three different sizes of SCC7 xenografts in BALB/c mice under US imaging guidance, where all the tumors were smaller than 1 cm (2.5~6 mm in lateral size). Immediately after USNG injection, notable contrast enhancement was observed repeatedly and reliably in the tumor tissues regardless of the size (Figure 4b and Movie S5), suggesting that the tumor microenvironment is indeed inflammatory and rich in H2O2 to induce the inflation of USNG into echogenic microbubbles. The resulting US molecular imaging signals visualized small tumors more clearly, which would provide higher precision for better tumor identification and size estimation. Having validated the diagnostic efficacy of USNG, we further explored its utility as a contrast agent by assessing the intraperitoneal toxicity at a high dosage level (60 mg/kg bodyweight) (Figure S11). The bodyweights of USNG-injected mice were shown to change negligibly compared to the PBS injected control mice. In blood test, red/white blood cell related factors were almost no difference between the experimental and control groups. Although differences were found in the values for monocyte, basophil and eosinophil, all the levels lie in


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the range of clinical standards for mice, along with no significant difference in liver functionrelated factors.40 In histologic examination, USNG-treated mice did not present any apparent lesions of hemorrhage, infarction, wide range of apoptosis/necrosis, structural damages, or immune cell infiltration in major visceral organs including heart, lung, kidney, spleen, and liver. All these safety data, along with the contrast-enhancing efficacy, suggest the potential utility of USNG for US molecular imaging.

CONCLUSIONS We have reported the development and application of a peroxamide-based ultrasound contrast agent (USNG) as a chemical system of H2O2-responsive gas (CO2) generation for ultrasound molecular imaging. The colloidal structure of USNG that intrinsically contains highly concentrated peroxamides and basic catalysts was found to serve as a nanoreactor for POCL reaction as well as a gas reservoir capable of nano-to-micro inflation, which resulted in the H2O2responsive US contrast enhancement in vitro and in vivo under a medical ultrasound frequency. In addition, the aliphatic peroxamide structure of USNG showed the long-term chemical stability against hydrolytic degradation as opposed to the rapid decay of a peroxalate ester derivative, ensuring the practical utility in biological media. We demonstrated that the H2O2-responsive US contrast enhancement by USNG is indeed operative for diagnostic imaging of inflammatory diseases in mouse models. By virtue of the material characteristics and imaging efficacy, as well as the biocompatibility of bPEI-peroxamide colloids, we envision that our efforts would provide a rational probe design strategy to drive further investigation and advancement of stimuliresponsive UCAs.


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dispersed peroxamide-concentrated nanogel colloid (USNG) by H2O2-responsive CO2 generation for US molecular imaging. (d) Peroxamide-crosslinked PEI bulk gel formed in methylene chloride. (e) FT-IR spectra of bPEI, peroxamide bulk gel and peroxamide nanogel. (f) CryoTEM image of USNG.


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-H2O2

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Figure 2. (a) H2O2-responsive CL images (top) and intensities (bottom) of USNG suspensions in PBS buffer at pH 7.4 and 4.5, as well as pH 7.4 reneutralized from pH 4.5 (pH 4.5 to pH 7.4). The error bars indicate the standard deviations for independent experiments (n = 5). (b)


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Fluorescence emission from the USNG suspensions at pH 4.5 and 7.4 without adding H2O2. (c) Chemical structures of a low-molecular-weight aliphatic peroxamide (DPO) and a peroxalate ester (CPPO). (d) The rise kinetic rates (kr) of DPO in the presence or absence of basic bPEI (0, 0.2 and 20 mg bPEI), along with that of CPPO alone. (e) The chemical stability of USNG against hydrolytic degradation in comparison to the CPPO nanoparticles, evaluated by monitoring the H2O2-responsive CL intensity of each aliquot sampled at the designated time points during the storage in PBS (pH 7.4) at 4 °C. The error bars indicate the standard deviations for independent experiments (n = 3).


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Before

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Inflammation

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Figure 4. Sonograms of acute liver inflammation and tumor in mouse models. (a) Normal and inflamed liver tissues before and 120 s after intrahepatic administration of USNG and the US intensities from vascular structures indicated in yellow in the sonograms. (b) SCC7 tumor tissues of three different sizes (2.5, 4, and 6 mm in lateral size) before and 5 s after intratumoral administration of USNG and the US intensities from the intratumoral region of the sonograms. The results are represented as the mean ± standard deviation (n = 3; *P < 0.02).


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METHODS Chemicals and reagents: All chemicals were purchased in Sigma-Aldrich and used without further purification. FlammaTM 675, Cy5.5 containing vinyl sulfone group, was obtained from BioActs Co. Ltd. in Korea. Synthesis of bPEI-peroxamide: bPEI-peroxamides were prepared by reaction between bPEI (Mn 0.6 k) and oxalyl chloride in reverse micelle (tween 80/cyclohexane) system. In detail, 1 g of tween 80 and 0.4 g of bPEI were added to 15 mL of cyclohexane under vigorous shaking to afford a homogeneous dispersion and 0.2 mL of oxalyl chloride was dropped to the dispersion. After vigorous shaking for 12 h at room temperature, the suspension mixture was poured into excess ethanol. The precipitated nanogels were purified by repetitive sequence (3 times) of centrifugation (10,000 rpm, 1 h), removal of supernatant and redispersion in ethanol under probe sonication. The purified nanogels were dried under vacuum. USNG preparation: 10 mg of the bPEI-peroxamides were dispersed in 1 mL of MilliQ-water or aq. Cy5.5-vinylsulfone solution (0.5 mg mL-1) with 1 =9 of PFH and 20 mg of Pluronic F68 by probe sonication. To remove unreacted Cy5.5-vinylsulfone, the sample was centrifuged for 1 h at 10,000 rpm and was resuspended in 1 mL of PBS (pH 7.4). The USNG samples were stored at 4 oC

prior to use.

Characterization: The chemical structures of bPEI-peroxamide and bulk peroxamide were characterized by FT-IR spectra (Perkin Elmer FT-IR system, Spectrum GX) at room temperature with 32 accumulations at a resolution of 4 cm-1. Particle size distribution of USNG was determined by a particle sizer (Zetasizer Nano ZS, Malvern, UK) and transmission electron microscopic (TEM) images with a CM30 electron microscope (FEI/Philips) operated at 200 kV.


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The morphology change of USNG with or without H2O2 was observed by cryogenic transmission electron microscopy (Cryo TEM, Tecnai G2 F20 Cryo, FEI, Netherlands). Determination of CO2 from bPEI-peroxamide in response of H2O2: Temperature-programmed desorption

(TPD;

ASAP2920;

Micromeritics)–mass

spectroscopy

(MS;HPR20;

Hiden

Analytical) experiments were performed to analyze gas generated by the reaction between dried bPEI-peroxamide and H2O2. For sample preparation, 400 mg of dried peroxamide nanogel was loaded with or without 2-3 drops of H2O2 (30 wt% in water) into a glass capillary of TPD-MS instrument. MS spectra were acquired under carrier gas flow (He, 30 cm3/min) at room temperature. In vitro CL imaging: To monitor the POCL reaction behavior of USNG, excess H2O2 (10 mM, 20 =92 or Milli-Q water as a negative control was added to 150 =9 of Cy5.5-vinylsulfone conjugated USNG and the mixtures were imaged with maximum 1 min acquisition time by an IVIS-200 imaging system (Xenogen, USA). In vitro US imaging: In vitro US images were obtained using Vevo770 (High-Resolution MicroImaging System, Visualsonics, Toronto, Canada), equipped with a RMV 706 probe. The study used an agar-gel phantom made by embedding a 500 =9 ependorf tube in the agar-gel (3%, w/v) and removing the tube after phantom gel was cooled. 300 =9 of USNG suspension was loaded into agar-gel phantom and imaged with 40 MHz of ultrasound. To evaluate the H2O2-induced enhancement of US intensity, excess H2O2 (final conc. 90 = 2 were added to the USNG suspension. The change of US intensity in each sample was measured up to 5 min, and the US intensity of the water control was subtracted from the sample intensity as the normalizing process.


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Preparation of acute liver injury model: All animal experiments were conducted according to the relevant laws and institutional guidelines of the Korea Institute of Science and Technology (KIST) and institutional committees. For preparation of acute liver inflammation model, BALB/c mice (male, 5 weeks of age; Orient Bio Inc., Korea) received an intravenous injection of 1 mg of P. acnes suspended in 100 =9 of PBS. After 7 d, the animals were given an intravenous injection of 1 = of lipopolysaccharide (LPS) in 200 =9 of PBS. To confirm the establishment of liver injury, blood test and histologic examination of the liver tissue were performed. At 12 h after LPS injection, the liver was enucleated to be fixed in 4% neutral buffered formalin. Livers of the PBS treated mice were also prepared as a control. The sections of the paraffin-embedded liver tissues (5 =

in thickness) were stained with hematoxylin and eosin for microscopic observation

(Olympus BX51; Tokyo, Japan). Blood samples from LPS-treated mice were collected at 12 h after LPS-injection and subjected to blood tests. In vivo US imaging of liver: At 12 h after LPS injection, the mice were anesthetized using isofluorane gas and abdominal hair was removed with depilatory cream. Mice were positioned in an animal pad (Vevo770 maintained at 37 °C) and the liver was imaged using a RMV706 probe. After obtaining pre-injection liver images, 200 =9 of USNG (30 mg/kg) were injected into the liver using a catheter syringe, and all the US images were recorded as a video file. The US images of the liver were obtained within 10 min. To compare the US enhancement by inflammation, US images of the normal liver were also obtained after injections of USNG suspension (30 mg/kg) using the same method. In vivo US imaging of tumor: Tumor xenografts were made by subcutaneous injection of a suspension of 1

107 SCC7 cells in RPMI1640 cell culture media into BALB/c nude mice

(male, 5 weeks of age; Orient Bio Inc., Korea). Two weeks after inoculation, the tumor-bearing


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mice were anesthetized using isofluorane gas and positioned in an animal pad. After obtaining pre-injection US images of solid tumor, 50 =9 of USNG suspension (30 mg/kg) was intratumorally injected and the tumor was imaged using a RMV706 probe. In vivo toxicity evaluation: 200 =9 of USNG at a high concentration (60 mg/kg) or PBS was intraperitoneally injected to BALB/c mice (~20 g bodyweight, male, 5 weeks of age; Orient Bio Inc., Korea). The bodyweight was measured at selected time points for 3 weeks. The blood samples were collected in 10 d of the USNG administration after anaesthetized with intraperitoneal injection of 0.5% pentobarbital sodium (0.01 mL g-1). The blood samples were analyzed by NEODIN Medical Institute (Korea).

ASSOCIATED CONTENTS Supporting Information Available: This material is available free of charge on the ACS publications website at DOI: Synthetic scheme of bPEI-oxamide nanogel; Hydrodynamic size of USNG in water; Stability of USNG in water over time; Zeta potential of bPEI-peroxamide nanogel and USNG; Chemiluminescence and fluorescence of USNG formulated with Cy5.5 labeled bPEI-peroxamide nanogel; POCL profiles of dipropyloxamide (DPO) and CPPO; Optical micrographs of microbubble generation of USNG and CPPO; Mass analysis of CO2 generated from bPEIperoxamide reacted with H2O2; in vitro US phantom images of USNG; Blood test and histologic analysis of acute liver injured mice; in vivo cytotoxicity evaluation of USNG.


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AUTHOR INFORMATION Corresponding Author * E-mail : [email protected] Author Contributions ‡These authors contributed equally. ORCID Jeongyun Heo: https://orcid.org/0000-0002-0507-9358 Chang-Keun Lim: https://orcid.org/0000-0003-1306-5770 Hyun Su Min: https://orcid.org/0000-0002-3158-2221 Kyung Eun Lee: https://orcid.org/0000-0001-5791-0046 Keunsoo Jeong: https://orcid.org/0000-0002-0227-2275 Young Hun Seo: https://orcid.org/0000-0002-8244-7791 Yong-Deok Lee: https://orcid.org/0000-0002-4408-3755 Ji Young Yhee: https://orcid.org/0000-0001-5992-2892 Kwangmeyung Kim: https://orcid.org/0000-0001-7919-188X Ick Chan Kwon: https://orcid.org/0000-0003-1272-707 Soo Young Park: https://orcid.org/0000-0002-2272-8524


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Sehoon Kim: https://orcid.org/0000-0002-8074-1006

ACKNOWLEDGMENT This work was supported by grants from the National Research Foundation of Korea (2017M3A9D8029942, and 2014M3C1A3054141), the Korea Health Industry Development Institute (HI15C1540), the Development of Platform Technology for Innovative Medical Measurements Program from Korea Research Institute of Standards and Science (KRISS-2018GP2018-0018), and the Intramural Research Program of KIST.


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