Radiofrequency-Sensitive Longitudinal Relaxation Tuning Strategy

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Cite This: ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Radiofrequency-Sensitive Longitudinal Relaxation Tuning Strategy Enabling the Visualization of Radiofrequency Ablation Intensified by Magnetic Composite Yan Fang,† Hong-Yan Li,† Hao-Hao Yin,† Shi-Hao Xu,† Wei-Wei Ren,† Shi-Si Ding,† Wei-Zhong Tang,*,‡ Li-Hua Xiang,† Rong Wu,*,§ Xin Guan,† and Kun Zhang*,†,‡ ACS Appl. Mater. Interfaces Downloaded from pubs.acs.org by UNIV OF ALABAMA BIRMINGHAM on 03/16/19. For personal use only.

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Department of Medical Ultrasound, Shanghai Tenth People’s Hospital, School of Medicine, Tongji University, 301 Yan-chang-zhong Road, Shanghai 200072, P. R. China ‡ A Guangxi Collaborative Innovation Center for Biomedicine, and Affiliated Tumor Hospital of Guangxi Medical University, Guangxi Medical University, 22 Shuang Yong Road, Nanning, Guangxi 530021, P. R. China § Department of Medical Ultrasound, Shanghai General Hospital, Shanghai Jiaotong University School of Medicine, 85 Wu-jin Road, Shanghai 200080, P. R. China S Supporting Information *

ABSTRACT: As a minimally invasive heat source, radiofrequency (RF) ablation still encounters potential damages to the surrounding normal tissues because of heat diffusion, high power, and long time. With a comprehensive understanding of the current state of the art on RF ablation, a magnetic composite using porous hollow iron oxide nanoparticles (HIONs) as carriers to load DL-menthol (DLM) has been engineered. This composite involves two protocols for enhancing RF ablation, that is, HION-mediated magnetothermal conversion in RF field and RF solidoid vaporation (RSV)-augmented inertial cavitation, respectively. A combined effect based on two protocols is found to improve energy transformation, and further, along with hydrophobic DLM-impeded heat diffusion, improve the energy utilization efficiency and significantly facilitate ex vivo and in vivo rf ablation. More significantly, in vitro and in vivo RSV processes and RSV-augmented inertial cavitation for rf ablation can be monitored by T1-weighted magnetic resonance imaging (MRI) via an RF-sensitive longitudinal relaxation tuning strategy because the RSV process can deplete DLM and make HION carriers permeable to water molecules, consequently improving the longitudinal relaxation rate of HIONs and enhancing T1-weighted MRI. Therefore, this RF-sensitive magnetic composite holds a great potential in lowering the power and time of RF ablation and improving its therapeutic safety. KEYWORDS: inertial cavitation, magnetothermal effect, longitudinal relaxation tuning, T1-weighted MRI monitoring, radiofrequency ablation

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no porous shells and cavities.13−15 Therefore, ensuring the therapeutic safety and improving the efficiency of RF ablation are of coequal significance and desirable, but both of the concerns are still challenging. With a deep and comprehensive understanding of RF ablation and oncology histopathology, three protocols are categorized to address the above two concerns, that is, suppressing blood-flow-mediated heat loss or diffusion, improving energy transformation from electromagnetic wave to phonon vibration, and monitoring RF ablation via noninvasive diagnosis tools. It has been extensively accepted

INTRODUCTION

Radiofrequency (RF) as a minimally invasive method has been widely used in clinics for heat ablation of malignant tumors.1−4 However, high ablation power and long ablation time are essential for acquiring excellent ablation outcomes in practical clinics, which, nevertheless, will probably give rise to severe burns to normal tissues surrounding lesions, especially for those minor lesions.5−8 To address this issue, some magnetic iron oxide particles were introduced to enhance the RF ablation efficiency via iron oxide-mediated magnetothermal conversion in the RF field.9−14 Despite improving the efficiency of RF ablation to some extent, magnetic iron oxide particle-assisted RF ablation protocol still suffers from some drawbacks, for example, blood flow-mediated heat loss, severe side effects, and failure of loading drug or medium because of © XXXX American Chemical Society

Received: February 6, 2019 Accepted: March 7, 2019 Published: March 7, 2019 A

DOI: 10.1021/acsami.9b02401 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

demonstrate this combined strategy for tremendously elevating the efficiency of RF ablation.

that the outcome of RF ablation primarily relies on the underlying principle, that is, energy transformation from electromagnetic wave to phonon vibration.11 Thus, improving the efficiency of energy transformation from electromagnetic wave to phonon vibration can serve as the primary pathway to improve RF ablation efficiency. Besides the aforementioned magnetic iron oxide-mediated magnetothermal effect, inertial cavitation that is usually explored in ultrasound theranostics can also be used to improve the efficiency of energy transformation from electromagnetic wave to phonon vibration.15−17 It has been confirmatively documented that cavitation-derived biophysical effects (e.g., microjets, shock waves, and local hyperpyrexia) can augment heat ablation efficiency including ultrasound, photothermal ablation, magnetothermal ablation, and RF ablation.15−19 As a paradigm, ethanol has been clinically approved to promote RF ablation via inertial cavitation caused by the rapid RF droplet vaporation (RDV) of ethanol.20−23 Unfortunately, akin to aforementioned iron oxide nanoparticles, ethanol failed to hamper the blood-flow-carried heat loss or diffusion because of its robust miscibility into blood, which in turn gave rise to injures to the surrounding normal tissues.24 In this regard, we established an RF solidoid vaporation (RSV) strategy for improving the efficiency of RF ablation via encapsulating solid DL-menthol (DLM) that can respond to heat and generate solid−liquid−gas (SLG) triphase transformation in poly(lactic-co-glycolic acid) (PLGA) carriers.15,17 In the RSV process, the introduced cavitation nuclei (i.e., DLM) could facilitate energy transformation and improve the efficiency of RF ablation on one hand, and it also could hinder heat diffusion because of the hydrophobic property of DLM on the other hand,25 which together contributed to the improved therapeutic safety. Nevertheless, no effective imaging means available for monitoring RF process remain an obstacle of clinical translation. Moreover, employing the aforementioned single protocol (e.g., iron oxide-mediated magnetothermal conversion or RSV or RDV-augmented inertial cavitation) to improve the efficiency of rf ablation is also not robust. Inspired by the previous work and accumulative experiences on RF ablation, we designed and engineered a distinctive RFsensitive magnetic composite using hollow iron oxide nanoparticles (HIONs) with porous shells as carriers to encapsulate DLM (abbreviated into DLM@HION). Because of the distinctive components and structures, this magnetic composite combines the aforementioned two protocols (i.e., HIONmediated magnetothermal conversion and DLM’s RSVaugmented inertial cavitation) for enhancing the rf ablation. In detail, HIONs assimilate the merit of previous magnetic iron oxide nanoparticles, that is, mediating the magnetothermal conversion in previous magnetothermal ablation under the alternating magnetic field,26 which, thus, is expected to enable the magnetothermal-effect-enhanced RF ablation in the RF field. Concurrently, the hollow cavity with porous shells can load or encapsulate DLM or other media, which is not equipped in previously reported iron oxide-based RF-enhanced ablation. As well, inertial cavitation mediated by RSV process of DLM in the RF field is expected to improve RF ablation, akin to our previously established PLGA-based RF enhancement agents.15 Thus, this magnetic composite is expected to respond to RF and improve the efficiency of RF ablation via the marriage of HION-mediated magnetothermal effect and DLM’s RSV-augmented inertial cavitation, as indicated in Scheme 1a. Ex vivo and in vivo experiments were carried out to

Scheme 1. Illustrations of the Combined Protocols (i.e., HION-Mediated Magnetothermal Conversion and DLM’s RSV-Augmented Inertial Cavitation) for Enhancing rf Ablation and rf-Sensitive Longitudinal Relaxation Tuning for Monitoring RF Process and RSV Process; (a) Schematic on Combined Enhancing Effect from HION-Mediated Magnetothermal Conversion and RSV-Enhanced Inertial Cavitation Effect Deriving from Encapsulated DLM; (b) Principle Schematic on RF-Sensitive Longitudinal Relaxation Tuning for Monitoring DLM-Mediated RSV and rf Ablation via Varying T1-Weighted MRI Signals before and after RF Ablation

More significantly, the RF ablation process can be monitored by tuning the longitudinal relaxation rate and measuring the variation of T1-weighted magnetic resonance imaging (MRI) signal that DLM-mediated RSV process causes. The adjoining permeability of HIONs to water can be enlarged after the RSV process of DLM upon exposure to RF field. This RSV process can further increase the number of bounded water molecules (q) and shorten the residence time (τm) of water molecules, consequently strengthening T1-weighted MRI signals (Scheme 1b) [i.e., IT1MRI (post-RF) > IT1MRI (pre-RF)], because the physical and chemical variations of T1-weighted MRI probes’ surface have been demonstrated to cause variations of the number of bounded water molecules on paramagenetic centers of metal ions and their exchange rate with outer-sphere water molecules.27,28 This variation of longitudinal relaxation rate can in turn be used to monitor the RSV-mediated RF ablation. In vitro and in vivo T1 -weighted MRI were explored to successfully demonstrate this magnetic composite (i.e., DLM@HION) for monitoring the RSV process, RSV-augmented inertial cavitation, and enhanced rf ablation, which promises the controllability of rf ablation via ceasing or continuing RF ablation according to the monitoring ablation outcome. This magnetic composite B


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Figure 1. Characterizations of magnetic composite (DLM@HION). (a−c) TEM (a), SEM (b), and bright-field (c) images of HIONs; (d,e) atom mapping of O (d) and Fe (e) in HIONs for determining their distributions; (f) X-ray diffraction pattern of HIONs, indicating the typical crystalline of Fe3O4 according to the PDF standard card (PDF#88-0315). (g,h) TG (g) and DSC (h) curves of HIONs and DLM@HION nanoparticles and the temperature in endothermic peak in DSC corresponds to that on the switching point of plateau.

Figure 2. Quantitative and qualitative inertial cavitation evaluation using ICD measurement apparatus and ultrasound imaging technology, respectively. (a) Schematic of the measurement apparatus available for quantitatively evaluating inertial cavitation via monitoring ICD value at different acoustic pressure, and samples were injected into the sample container (3 × 3 × 3 cm) via the extension tube; (b) quantitative ICD values of two samples (i.e., HION and DLM@HION) at room temperature (25 °C) and 45 °C, respectively. (c) Qualitative evaluation on SLG triphase transition via the RSV process using BFI and CHI ultrasound imaging modes before and after RF heating.

magnetothermal transition.26 More significantly, rich porous channels and hollow interiors can reserve capacity enough to accommodate various molecules, endowing HIONs with more functions. In an attempt to fabricate porous HIONs, a simple hydrothermal synthesis method was adopted herein.18,26 It is clearly found that the as-prepared HIONs feature hollow structure and porous shell, as evidenced in transmission electron microscopy (TEM) images (Figures 1a and S1) and scanning electron microscopy (SEM) images (Figures 1b,c and S2). The F and O atoms are uniformly distributed in HIONs, as shown in Figure 1d,e. No evident hollow structure in atomic

combines enhanced RF ablation arising from the combined effect of the aforementioned two protocols with MRI monitoring stemming from RF-sensitive longitudinal relaxation tuning, which maximizes the RF ablation efficiency and therapeutic safety, thus holding a great potential in future clinical translation.

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RESULTS AND DISCUSSION Synthesis of DLM@HION. As one subtype of magnetic iron oxide nanoparticles, porous HIONs also exhibit high saturated magnetization intensity, enabling the highly efficient C


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In particular, the SLG process of DLM in DLM@HION can be qualitatively monitored by ultrasound imaging technology,15,17 and two ultrasound imaging modes, that is, B fundamental imaging (BFI) and contrast harmonic imaging (CHI) that only can reflect the two-fold harmonic signals produced by bubbles, were employed.32 It is clearly found that no evident contrast enhancement is found in two comparison groups [i.e., phosphate-buffered saline (PBS) and HION] no matter what kind of ultrasound imaging mode is used (Figure 2c). On the contrary, lots of DLM bubbles are observed under CHI mode in comparison with HION alone, resulting in the significantly improved contrast in DLM@HION after RF ablation. These results confirmatively suggest such a magnetic composite can respond to RF heating, produce DLM bubbles via the RSV process, and further result in inertial cavitation, which is expected to improve the energy transformation and enhance the following RF ablation along with HION carriermediated magnetothermal conversion. In Vitro T1-Weighted MRI for Monitoring RSV via RFSensitive Longitudinal Relaxation Tuning. According to the degree of RF ablation, triggering or ceasing RF ablation at the right time is another solution to guarantee the therapeutic safety. To this end, monitoring the degree of RF ablation is necessary. Pre-existing imaging technologies (i.e., ultrasound contrast imaging) in clinics are not available for monitoring the RSV process because it is hard to distinguish whether the increased contrast of ultrasonic images in ablated zone resulted from the hardness increase of ablated tissues or uncontrollable RSV-mediated bubbles’ production.33,34 Besides ultrasound imaging-guided RF ablation, MRI as a quantitative method that is usually designed for detecting tumor biomarkers27,35 has been also extensively employed to monitor the RF process when using iron oxide nanoparticles to enhance RF ablation,10,36 akin to other MRI-monitoring thermal ablation technologies (e.g., high-intensity focused ultrasound).37,38 However, almost all previous MRI monitoring cases in RF ablation using iron oxide particles focus on T2-weighted MRI. In principle, T2-weighted MRI of iron oxide-based probe is inapplicable for therapeutic monitoring because of the potential inversion of T2-weighted MRI signal and poor sensitivity to variations of surface and surrounding features of iron oxide probes that RF field and RF heat cause. Herein, despite featuring T2-weighted MRI ability because of the large r2 (186.39 mM−1 s−1)/r1 (0.696 mM−1 s−1) value (i.e., 267.8) (Figure S7), the T2-weighted MRI of DLM@ HION nanoparticles fails to monitor the inertial cavitation and RF process. It can be attributed that the T2-weighted MRI signal inversion at a dose far lower than the therapeutic dose occurs (Figure S8), and RF-mediated physical or chemical or biological variations fail to vary T2-weighted MRI, as evidenced by no evident difference of T2-weighted MRI between DLM@ HION (r2 = 172.74 mM−1 s−1, Figure S9) and HION (r2 = 186.39 mM−1 s−1, Figure S7b). It has been reported that iron oxide could also serve as a probe for T1-weighted MRI.39 To address the two issues that T2-weighted MRI encountered, the RF-sensitive longitudinal relaxation of DLM@HION was evaluated before and after RF irradiation for monitoring DLM-mediated inertial cavitation and RF ablation. It is found that the brighter contrast representing T1-weighted MRI signal intensity after RF irradiation is observed at any given Fe molar concentration, as exhibited in Figure 3a. This phenomenon can be explained in Scheme 1b wherein hydrophobic DLM occupied the interior cavity and porous

mapping images is attributed to the thick shell and irregular porous channels in the interior of HIONs in the low-power field. HIONs exhibit a typical Fe3O4 crystalline according to the standard card (PDF#88-0315), as shown in Figure 1f. The well-defined HIONs with a hydrated dynamic diameter of 363 nm feature a negative surface zeta potential (i.e., −38.2 mV) probably because of ethylene glycol (EG) molecules with a 4% content in HIONs (Figure 1g), as shown in Figure S3. The negatively charged surface is preferable to the positively charged surface because the negative surface potential was more beneficial for inhibiting particle agglomeration.29 Depending on the large capacity because of large pores and hollow interiors in HION carriers (Figure S4), DLM can be easily encapsulated by HIONs with an encapsulation efficiency of cal. 16.47% via a well-established solvent replacement method,25 because HIONs are hydrophilic. Thermogravimetric (TG) analysis and differential scanning calorimetry (DSC) were employed to demonstrate the successful loading of DLM and determine the loading amount of DLM in DLM@ HION.25 With analyzing the temperature-dependent TG curves of HIONs and DLM@HION, both of which probably contains EG molecules, the loading amount of DLM in DLM@HION is calculated to be 14.06%, as shown in Figure 1g,h. Identical to other Fe3O4 nanoparticles,30,31 the asprepared HIONs are also superparamagnetic, as evidenced in the magnetic hysteresis loop (Figure S5), which determines that HIONs are equipped with the robust magnetic−thermal transition property.26 As expected, the time-dependent temperature elevation profile and infrared (IR) images induced by the magnetothermal conversion of HIONs with varied concentration suggest the excellent magnetic−thermal transition ability of HIONs under the alternating magnetic field (Figure S6), which lays a robust guarantee for heating and enhanced hyperpyrexia ablation in the electromagnetic field of RF. Inertial Cavitation Measurement of DLM@HION. It has been confirmed that DLM can respond to RF or high intensityfocused ultrasound to generate bubbles via the SLG process15,25 and further continuously produce inertial cavitation.15 To demonstrate whether DLM endows DLM@ HION with the identical effect on RF ablation, we quantitatively explored the cavitation effect arising from heattriggering explosion of DLM bubbles. The apparatus that is available for measuring the inertial cavitation dose (ICD) is found in Figure 2a, wherein the temperature of water-filled container can be tuned via directly heating, and a 15 MHz transducer can receive ultrasound signals with a wide range of frequencies that is produced by inertial cavitation. Results show neglectable ICD at room temperature in both HION and DLM@HION, indicating no occurrence of inertial cavitation, as evidenced in Figure 2b. In contrast, once the surrounding temperature is elevated to 45 °C that is above the solid−liquid triphase transition point (i.e., 42 °C) of solid DLM, significantly increased ICD values within the measuring window of acoustic pressure are observed in DLM@HION, whereas no evident ICD increase occurs to HIONs alone. This intriguing phenomenon sufficiently demonstrates the presence of inertial cavitation deriving from liquid DLM volatilizationderived bubbles’ explosion via the hyperpyrexia-accelerated SLG process. This result lays a solid foundation to enable RFsensitive inertial cavitation of DLM in DLM@HION for enhancing RF ablation. D


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irradiation, resulting in the reduced number of water molecules bounded with HION (q). Moreover, this hydrophobic DLMmediated insulation also decreased the solvent exchange rate (kex) of bounded water molecules to free water molecules and prolonged the residence time (τm) of water molecules on HION, that is, the reciprocal of the exchange rate (kex).28 On exposure to RF irradiation, the SLG triphase transition of DLM is available for impairing the insulation, and HION carriers were liberated without wrapping by hydrophobic DLM, consequently resulting in q increase, kex elevation, and τm shortening. Both q increase and τm shortening will further result in the increase of longitudinal relaxation rate (1/T1) that is obtained according to the following inner-sphere relaxation formula.27,28 qPm 1 = T1 T1m + τm

(1)

where Pm is the mole fraction of Fe, and T1m is the relaxation time of water proton spin. As expected, T1-weighted MRI is indeed strengthened and the T1 signal intensity (i.e., brightness) of IT1MRI (post-RF) at any given Fe molar concentration is larger than that of IT1MRI (pre-RF), that is, IT1MRI (post-RF) > IT1MRI (pre-RF), as evidenced in Figure 3a. The quantitative contrast value representing the T1-weighted MRI signal intensity is also obtained in Figure 3b. It is clearly found that all of the concentration-dependent contrast values of T1-weighted MRI in post-RF group are larger than those in pre-RF at the identical Fe molar concentration, resulting in a larger slope value of contrast. Besides contrast, longitudinal relaxivity (r1) can also confirm it because r1 (post-RF) value is much larger than r1 (pre-RF), that is, 0.599 mM−1 s−1 versus 0.256 mM−1

Figure 3. Evaluations on in vitro RF-sensitive T1-weighted MRI of DLM@HION before and after RF irradiation. (a) In vitro MRI images of DLM@HION as a function of Fe molar concentration before and after RF irradiation (5 W20 s), and the region of interest is circled by yellow dotted line; (b) T1-weighted signal intensity variation of DLM@HION before and after RF irradiation that is obtained via measuring the contrast values of MRI images (a); and (c) longitudinal relaxation of DLM@HIONs, a function of Fe molar concentration before and after RF irradiation (5 W20 s).

channels of HION carriers in DLM@HION and insulated DLM-wrapped HION carriers from water molecules before RF

Figure 4. Ex vivo evaluations on combined RF ablation enhancement on pork liver based on two principles, that is, HION-mediated magnetothermal transition and DLM-enhanced inertial cavitation. (a) Schematic on the ex vivo RF ablation on pork liver (n = 3); (b) ultrasound images of pork liver treated with different samples before and after RF irradiation (5 W20 s), and in RF alone, PBS was injected, whereas in HION + RF and DLM@HION + RF, HION and DLM@HION were injected, respectively. (c) Digital photos of ablated tissues after treatment with different groups, that is, RF alone, HION + RF, and DLM@HION + RF, wherein ablated zones are circled by ellipses with yellow dotted line and (d) quantitative ablated volume on pork liver after treatment with different groups, that is, RF alone, HION + RF, and DLM@HION + RF; notes * and ** represent P < 0.05 and 0.01, respectively. E


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Figure 5. In vivo enhanced RF ablation against tumor based on the combined effect of HION-mediated magnetothermal transition and DLMenhanced inertial cavitation under RF-sensitive MRI monitoring. (a) Schematic on in vivo RF ablation on 4T1 xenografted tumor subcutaneously implanted on nude mice with certain RF parameter (5 W15 s), wherein the intratumoral injection manner was employed; (b) digital photos of ablated tumor tissues after treatment with different groups, that is, RF alone, HION + RF, and DLM@HION + RF, wherein ablated zones are circled by ellipses with yellow dotted line; and (c) quantitative ablated volume on 4T1 tumor after treatment with different groups, that is, RF alone, HION + RF, and DLM@HION + RF; notes “*”, “**”, and “***” represent P < 0.05, 0.01, and 0.001, respectively. (d) In vivo T1-weighted MRI images of 4T1 tumor subcutaneously implanted on nude mice before and after RF irradiation, and the mean contrast value is obtained via the RadiAnt Viewer software. (e) Pathological diagnosis (i.e., H&E) and immunohistological examinations (i.e., PCNA and TUNNEL) of tumor slices after treatment with different groups, that is, control, RF alone, HION + RF, and DLM@HION + RF, and yellow arrows indicate the ablated boundary. (f) Western blot images of tumor tissues after treatment with different groups, that is, control, RF alone, HION + RF, and DLM@HION + RF.

s−1. Therefore, this RF-responsive longitudinal relaxation tuning can monitor the RSV process of DLM in DLM@ HION, which enables the monitoring of enhanced RF ablation by RSV-augmented inertial cavitation. Noticeably, the structure and crystalline nature of HION in DLM@HION after RF ablation are hardly affected, suggesting excellent thermal stability of HION carriers (Figure S10). As well, slight variations of hydrated particle size and surface zeta potential of DLM@HION after RF ablation further demonstrate the occurrence of RF-induced DLM phase transition (Figure S11). Ex Vivo Enhanced RF Ablation Using the Combined Strategy of HION-Mediated Magnetothermal Conversion and DLM’s RSV-Augmented Inertial Cavitation. After successfully demonstrating RSV-augmented inertial cavitation, the ability of the enhanced inertial cavitation in RF field to augment RF ablation was explored on ex vivo pork liver along with HION-mediated magnetothermal conversion. The schematic of the ex vivo RF ablation experiment on pork liver is shown in Figure 4a, and the BFI mode was first employed to preliminarily determine ablation outcomes against ex vivo pork livers treated with different groups. No evident ultrasonic contrast increase of ex vivo ablated zone in pork liver after RF ablation is found in either RF alone or

HION + RF, as shown in Figure 4b. In contrast, a significantly improved contrast is observed in the group of DLM@HION + RF after RF irradiation for 20 s under 5 W, implying that DLM@HION + RF produces a much larger ablation volume than RF alone and HION + RF, according to previous experiences.25 To further confirm it, the ablated tissues were sliced and directly observed. In comparison with RF alone, HION + RF harvests a much larger ablation zone, successfully demonstrating the occurrence of HION-mediated magnetothermal conversion for enhancing RF ablation, as evidenced in Figure 4c. In particular, after encapsulating DLM in HION, DLM’s RSV-augmented inertial cavitation further enhances RF ablation, consequently resulting in the largest ablation zone in the pork liver treated with DLM@HION after RF ablation. Accordingly, the quantitative ablation volume was also calculated, and the ablation volume (29.7 mm3) in HION + RF is five times larger than that in RF alone (5.1 mm3) because of the HION-mediated magnetothermal conversion for enhancing RF ablation, as shown in Figure 4d. Furthermore, DLM@HION + RF harvests the largest ablation volume (66.5 mm3) with a 124% increase in relation to HION + RF, which can be attributed to the DLM inertial cavitation. Similar results can be also obtained on pork liver using another set of RF F


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observed in comparison with pre-RF, and accordingly the quantitative mean signal intensity increases from 279.22 (preRF) to 335.13 (post-RF), as can be found in Figure 5d. This result indicates that the occurrence of in vivo rf-triggered SLG process of DLM-recovered T1-weighted MRI ability of HION, validating the potential of the RF-sensitive longitudinal relaxation tuning strategy in monitoring in vivo RSV process. Moreover, no evident variation of T1-weighted MRI using DLM@PLGA that shares the same DLM dose with DLM@ HION before and after RF irradiation suggests that ablated zone fails to influence T1-weighted MRI signal (Figure S16), even though DLM@PLGA receives a significantly intensified ablated volume (Figure S17) via DLM-mediated continuous cavitation.15 This result further validates that in vivo RFtriggered SLG process of DLM is responsible for varying T1weighted MRI of DLM@HION, which enables the monitoring of in vivo RSV process. According to the in vivo comparison between the two increased magnitudes of ablated volumes from RF alone to HION + RF (ΔV = 14.1 mm3) and from HION + RF to DLM@HION + RF (ΔV = 47 mm3), the increased magnitude from HION + RF to DLM@HION + RF is dominant. This result suggests that RSV-augmented inertial cavitation is responsible for the substantially enhanced ablation volume, that is, the RSV progression primarily manipulates the extent of RF ablation. As well, a neglectable gap of ablated volume between DLM@HION and DLM@PLGA is observed (Figure S17), suggesting that RSV-augmented inertial cavitation is responsible for the substantially enhanced ablated volume. Therefore, it is feasible that the progression extent of RSV that can be monitored via this RF-sensitive longitudinal relaxation tuning strategy can be taken as a reference to decide to continue or cease RF ablation so as to assure the therapeutic safety. Molecular biological examinations were carried out to evaluate the mechanism of RF ablation using such a magnetic composite. Hematoxylin−eosin (H&E)-stained microscopic images of tumor slices treated with DLM@HION treatment in the presence of RF show the largest regions of significantly reduced cell density, the disappearance of cell structure, lysed cell membranes, and ruptured nuclei in the ablated zone, as shown in the upper row of Figure 5e. Moreover, proliferating cell nuclear antigen (PCNA)-based immunohistochemical staining and TUNNEL immunohistochemical staining were employed to label newly proliferative cells and apoptotic cells, respectively. It is clearly found that besides the sparsest cells, the group of DLM@HION + RF also results in the lowest proportion of proliferative cells (negative) in PCNA-stained microscopic images and the largest proportion of apoptotic cells (positive) in tunnel-stained microscopic images, respectively, as shown in Figure 5e. These results suggest that DLM@HION + RF killed 4T1 cells via inducing cell necrosis and apoptosis and simultaneously inhibited cell proliferation. Furthermore, western blot was used to further investigate the deep enhancement mechanism of this combined RF ablation protocol. Results show that the deep mechanism of combined RF ablation follows the upregulation pathways of several proapoptotic proteins (i.e., Bax, caspase 3, HSP70, and P53) and the downregulation pathway of antiapoptotic protein (e.g., Bcl-2), as shown in Figures 5f and S18. Although this MRI-monitoring RF ablation with combined RF enhancement strategy using such a magnetic composite can substantially improve the therapeutic safety and efficiency of RF ablation and inhibit tumor growth, tumor recurrence is still

parameters (i.e., 5 W10 s) (Figure S12). These results sufficiently validate that the marriage of two protocols, that is, HION-mediated magnetothermal conversion and DLM’s RSVaugmented inertial cavitation, cooperatively contributed to the intensified RF ablation. In Vivo Enhanced rf Ablation Using the Combined Effect of HION-Mediated Magnetothermal Conversion and DLM’s RSV-Augmented Inertial Cavitation under RF-Sensitive MRI Monitoring. After evaluations on ex vivo RF ablation, the in vivo RF ablation on 4T1 xenografted tumor subcutaneously implanted on nude mice was explored, and the experimental schematic is exhibited in Figure 5a. The high colloidal stability of DLM@HION nanoparticles guarantees the injectability of DLM@HION dispersions (Figure S13). Akin to previous cases, for example, ethanol or other iron oxide particles in RF ablation,10,23 immunotherapy,40 and hydrogelmediated drug delivery,41 intratumoral administering manner that also enables particles to pervade throughout the whole tumor was employed.42 Herein, ultrasound imaging under the BFI mode was employed to preliminarily evaluate the outcome of RF ablation. After RF ablation, the largest illumination zone that promises the largest ablation volume results from the group of DLM@HION + RF (Figure S14). Contributed by HION-mediated magnetothermal conversion, HIONs are endowed with a robust capability of enhancing RF ablation and thus generate a much larger ablation volume (14.1 mm3) in comparison with RF alone (0 mm3), which is similar to result, as indicated in Figure 5b,c. In particular, after further assistance with DLM’s RSV-augmented inertial cavitation, DLM@HION receives the largest ablation volume (61.1 mm3) that is four fold larger than HION after RF irradiation. These results sufficiently demonstrate the in vivo feasibility of this combined effect based on HION-mediated magnetothermal conversion and DLM RSV-augmented inertial cavitation for enhancing RF ablation. To acquire a deep understanding of the improved energy utilization efficiency for enhancing RF ablation, in vivo temperature variation of 4T1 tumors was recorded during RF irradiation. As can be found in Figure S15, the introduction of HIONs elevates the temperature from 47.3 °C (Figure S15a1,a2) to 56.9 °C (Figure S15b1,b2), suggesting that the HION-mediated magnetothermal conversion improves the energy utilization efficiency of RF-induced heat. Compared to HIONs alone, the magnetic composite that loads DLM (i.e., DLM@HION) further facilitates the elevation of maximum temperature from 56.9 °C (Figure S15b1,b2) to 90 °C (Figure S15c1,c2). This phenomenon is primarily attributed to the tremendously improved energy transformation from electromagnetic wave to phonon vibration that HION-mediated magnetothermal conversion and DLM’s RSV-augmented inertial cavitation cooperatively brought about. In addition, the hydrophobic DLM also serves as thermal-insulation media to inhibit heat diffusion and blood-flow-carrying heat loss and further improve energy utilization efficiency for enhancing RF ablation. As stated in vitro experiment, the principle of RF-sensitive longitudinal relaxation tuning for RSV monitoring lies in the variation of q and τm before and after RF irradiation. To explore the in vivo applicability of this strategy, the T1weighted MRI of 4T1 xenografted tumor subcutaneously implanted on nude mice treated with intratumoral injection of DLM@HION before and after RF irradiation was carried out. A brighter contrast at the site of tumor after RF irradiation is G


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loaded nanoparticles were collected via magnetic separation using magnet and then washed and sonicated with deionized water for use. Evaluations on Ex Vivo RF Ablation of Porcine Liver Tissues. The porcine liver tissues in vitro (3 × 3 × 2 cm3) were prepared and placed on the electrode board. Afterward, 0.1 mL of PBS, HION (5 mg/mL), and DLM@HION (5.8 mg/mL) dispersions in degassed PBS was injected into the middle of the porcine liver tissues, respectively. After rf (STARmed) irradiation (5 W) for 10 or 20 s with the water circulation system, the ablated porcine liver was dissected, and then the slices of the maximum ablation area were selected to calculate the ablation volumes. As well, before and after RF irradiation, B-mode ultrasound images were obtained by SuperSonic Imagine SWE system (probe: SL 15-4, MI = 1.5). The volume formula (V, mm3) of ablated tissues is shown as follows

unavoidable because of the presence of residual unablated tumor tissues (Figure S19). Therefore, future work will focus on the combined therapies, for example, chemotherapy and RF ablation, immunotherapy and RF ablation, and other emerging therapeutic protocols.43−51 Although it has been validated that the carriers, that is, porous HIONs, exhibited a high biocompatibility and could be excreted out of the body,26 we evaluated this biocompatibility of DLM@HION again. Compared to control, no evident damage in the group of DLM@HION + RF is observed, as shown in Figure S20, validating the potential therapeutic safety using such a magnetic composite. Neglectable variations of serum biochemical level between experimental group and control group reflect the biosafety and biocompatibility of this magnetic composite, as indicated in Figure S21.

V = L × W 2 × π /6

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where L and W represent the maximum length (unit: mm) and width (unit: mm) of ablated area, respectively. In Vitro MR Imaging. To evaluate in vitro T1-weighted MR imaging, 1 mL of DLM@HION dispersions at different Fe concentrations (0−0.5 mM) was added in Eppendorf tubes (2 mL volume). Before and after RF under 50 W for 20 s, T1-weighted MRI scanning was carried out on United Imaging (uMR 570, 1.5 T). As well, molar concentration-dependent T1-weighted MRI of HIONs was also implemented, and the pulse sequence used was FSE-T1WI with the following parameters: TR = 400, TE = 20, slice thickness = 2.00 mm, matrix = 180 × 180, and Acq (NEX) = 1. The T2-weighted MRI experiments of HIONs and DLM@HION at different Fe concentrations (0−0.05 mM) were also implemented, and the pulse sequence used was a T2-weighted fast-recovery fast spin-echo sequence with the following parameters: TR = 2800 ms, slice thickness = 2.0, TE = 57 ms, and echo length = 10 ms. In Vivo MR Imaging. To evaluate in vivo T1-weighted MR imaging, 0.1 mL of DLM@HION (5.8 mg/mL) dispersion in PBS was intratumorally injected into 4T1 tumor-bearing nude mice. MR imaging of tumor-bearing mice was gained before and after RF irradiation (5 W) for 15 s with the water circulation system on. The in vivo T1-weighted MRI experiment was carried out on Siemens Verio 3.0T MRI scanner with a scanning sequence: T1FLAIR-TRA, TR: 9.8 ms, and TE: 3.8 ms. As well, DLM@PLGA that shared the identical DLM dose with DLM@HION was also used as a control, and before and after RF ablation, the T1-weighted MRI was implemented. All of the procedures and parameters were the same with those of DLM@ HION. Evaluations on In Vivo RF Ablation of Tumor by DLM@ HION. Two sets of experiments were carried out for evaluating RF ablation and inhibited tumor growth, and the detailed details were depicted as follows: In Vivo Evaluations on RF Ablation. Twelve tumor-bearing female nude mice (17−22 g) were randomly divided into four groups (n = 3): (1) control group (no intratumoral injection and no RF irradiation), (2) RF alone (intratumorally injected with 0.1 mL PBS), (3) HION + RF (intratumorally injected with 0.1 mL of HIONs dispersion in PBS), and (4) DLM@HION + RF (intratumorally injected with 0.1 mL of DLM@ HION dispersion in PBS). In other three experimental groups (2−4), after intratumorally injecting 0.1 mL of PBS, HION (5 mg/mL) and DLM@HION (5.8 mg/mL) dispersions in PBS, RF ablation was carried out by a certain power (5 W) for 15 s with the water circulation system. During RF irradiation process, IR thermal imaging was captured by an IR imaging instrument (FLIR A325SC camera, USA) to record the timedependent temperature variation and capture thermal images at different time points. As well, before and after RF irradiation, B-mode ultrasound images were obtained by SuperSonic Imagine SWE system (probe: SL 15-4, MI = 1.5). The ablated tumors in other three experimental groups (2−4) and untreated tumors in the control group (1) were collected after 4 h post-RF, and the tumor slices were isolated and stained with 2,3,5-triphenyltetrazolium chloride (TTC) for observing the ablated areas. The maximum length (L, mm) and width (W, mm) of coagulated tissues were measured by using a

CONCLUSIONS In summary, a combined strategy for enhancing v ablation and improving therapeutic safety was established, and RF-sensitive magnetic composite using porous HIONs as carriers to encapsulate DLM was prepared to validate this combined strategy. The as-prepared magnetic composite was demonstrated to significantly reinforce ex vivo and in vivo RF ablation via the improved energy utilization efficiency enabled by the combined effect of HION-mediated magnetothermal conversion, DLM’s RSV-augmented inertial cavitation, and hydrophobic DLM-inhibited heat diffusion and blood-flowcarried heat loss. Deep investigations demonstrated that this combined effect for enhancing RF ablation with increased therapeutic safety followed proapoptosis/necrosis and antiproliferation pathways via upregulations of several proapoptotic proteins and downregulation of antiapoptotic protein, respectively. More significantly, RF-mediated RSV process of DLM in DLM@HION could deplete DLM to make more DLM-wrapping HIONs liberated and recover the T1-weighted MRI ability of HION via shortening τm and increasing q. Therefore, such an RF-sensitive longitudinal relaxation tuning strategy successfully monitored DLM-mediated in vitro and in vivo RSV process. Furthermore, it has been demonstrated that DLM’s RSV-augmented inertial cavitation primarily contributed to the enhanced RF ablation. Thus, this RF-sensitive longitudinal relaxation tuning strategy can further monitor RF ablation, which is beneficial for manipulating the RF ablation via ceasing or continuing RF ablation according to the monitored outcome and safety. This RF-sensitive magnetic composite for MRI-monitoring tumor ablation via the combined effect based on augmented cavitation, magnetothermal conversion, and inhibited heat loss paves a new avenue to lower the power and time of RF ablation and thus can drive the development of RF ablation against oncology.

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MATERIALS AND METHODS

Synthesis of DLM-Encapsulated Magnetic Composite (DLM@HION). HIONs (1 g) were first dispersed in methanol (2 mL) solvent by ultrasonic irradiation. Afterward, the HION dispersion was added into DLM solution (1 g, 70 °C) and then was stirred mechanically under the same condition of temperature for 5 h. With the gradual evaporation of methanol, DLM entered the hollow interior of HION. Subsequently, hot water was added. Immediately afterward, a violent stirring for another 10 min was carried out, and then static phase separation including aqueous phase containing DLM@HION and oil phase made of residual liquid DLM were gradually generated after 10 min. Ultimately, the resultant DLMH


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ACS Applied Materials & Interfaces caliper, and the ablated volume was calculated according to the formula: L × W2 ×π/6. Tumor issues without TTC staining were used for PCNA and tunnel immumohistochemical staining, H&E staining, and western blot assay. As well, another control, that is, DLM@PLGA, was used, wherein the DLM dose is identical with that in DLM@HION. Inhibited Tumor Growth. Twenty-four tumor-bearing female nude mice (17−22 g) were separated into four groups (n = 6) randomly: (1) control group (no intratumoral injection and no RF irradiation); (2) RF alone (intratumorally injected with 0.1 mL of PBS and exposed to RF irradiation at a power density of 5 W for 15 s); (3) HION + RF (intratumorally injected with 0.1 mL of HIONs dispersion in PBS at the dose of 5 mg HION per milliliter and exposed to RF irradiation at a power density of 5 W for 15 s); and (4) DLM@ HION + RF (intratumorally injected with 0.1 mL DLM@ HION dispersion in PBS at the dose of 5 mg HION per milliliter and exposed to RF irradiation at a power density of 5 W for 15 s). After different treatments, the weight and tumor volume of each mouse were obtained at several certain days, and the formula, that is, L × W2/2, was used to calculate the tumor volume, wherein L and W represent the measured length and width of tumor, respectively. After 18 days, several nude mice in each group were dissected, and the major organs (heart, lung, liver, kidney, and spleen) were obtained for H&E staining to analyze the change of pathological histology. During the period of breeding, at certain days, the serum biochemical measurements were implemented. Statistical Analysis. All of the experiments were performed in triplicate. The statistical significance between two groups was analyzed by the Student’s two-tailed t-test through SPS 22.0. Single, double, and triple asterisks represent P ≤ 0.05, 0.01, and 0.001, respectively.

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Opening Project of State Key Laboratory of High Performance Ceramics and Superfine Microstructure (SKL201811SIC).

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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.9b02401. Materials, additional experimental details, and supplementary figures (PDF)

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REFERENCES

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (W.-Z.T.). *E-mail: [email protected] (R.W.). *E-mail: [email protected] (K.Z.). ORCID

Kun Zhang: 0000-0002-6971-1164 Author Contributions

Y.F., and H.-Y.L. contributed equally to this work. K.Z. conceived and designed the project. Y.F., H.-Y.L., H.-H.Y., S.H.X., W.-W.R., S.-S.D., L.-H.X., and X.G. performed the experiment. R.W., W.-Z.T., and K.Z. monitored the project. K.Z. analyzed the data and wrote the paper. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge the support from the National Natural Science Foundation of China (grant nos. 81771836, 81501473, and 81671699), Guangxi Collaborative Innovation Center of Biomedicine (grant no. GCICB-SR-201703), Fostering Project of Shanghai Municipal Commission of Health and Family Planning for Excellent Young Medical Scholars (grant no. 2018YQ31), the Opening Project of Guangxi Key Laboratory of Bio-targeting Theranostics (GXSWBX201801), and the I


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Radiofrequency-Sensitive Longitudinal Relaxation Tuning Strategy

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