Continuous Cavitation Designed for Enhancing Radiofrequency Ablation via a Special Radiofrequency Solidoid Vaporization Process Kun Zhang,†,§ Pei Li,† Hangrong Chen,‡ Xiaowan Bo,† Xiaolong Li,† and Huixiong Xu*,†,§ †
Department of Medical Ultrasound, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, 301 Yan-chang-zhong Road, Shanghai 200072, P. R. China ‡ State Key Laboratory of High Performance Ceramic and Superfine Microstructures, Shanghai Institute of Ceramics, Chinese Academy of Science, 1295 Ding-Xi Road, Shanghai 200050, P. R. China § Ultrasound Research and Education Institute, Tongji University School of Medicine, 301 Yan-chang-zhong Road, Shanghai 200072, P. R. China S Supporting Information *
ABSTRACT: Lowering power output and radiation time during radiofrequency (RF) ablation is still a challenge. Although it is documented that metal-based magnetothermal conversion and microbubbles-based inertial cavitation have been tried to overcome above issues, disputed toxicity and poor magnetothermal conversion efficiency for metal-based nanoparticles and violent but transient cavitation for microbubbles are inappropriate for enhancing RF ablation. In this report, a strategy, i.e., continuous cavitation, has been proposed, and solid menthol-encapsulated poly lactide-glycolide acid (PLGA) nanocapsules have been constructed, as a proof of concept, to validate the role of such a continuous cavitation principle in continuously enhancing RF ablation. The synthesized PLGA-based nanocapsules can respond to RF to generate menthol bubbles via distinctive radiofrequency solidoid vaporization (RSV) process, meanwhile significantly enhance ultrasound imaging for HeLa solid tumor, and further facilitate RF ablation via the continuous cavitation, as systematically demonstrated both in vitro and in vivo. Importantly, this RSV strategy can overcome drawbacks and limitations of acoustic droplet vaporization (ADV) and optical droplet vaporization (ODV), and will probably find broad applications in further cancer theranostics. KEYWORDS: continuous cavitation, radiofrequency ablation, triphase transformation, radiofrequency solidoid vaporization, poly lactide-glycolide acid (PLGA) nanocapsules
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for enhancing high intensity focused ultrasound (HIFU) ablation via some special physical factors,11,12 e.g., shear stress, shock waves, local hyperpyrexia and microjets.9,13 Therefore, bubblesinduced cavitation, in principle, is expected to be a candidate strategy to enhance RF ablation. However, the common theranostic systems that generated cavitation, microbubbles, are still limited by the inherent paradox between large-sized bubbles for excellent imaging performance and small-sized bubbles for avoiding capture by the reticuloendothelial system (RES).12,14 Very recently, smart theranostic agents have been engineered, wherein encapsulated liquid fluorocarbons could evaporate into large micrometer-sized bubbles upon external triggering, and afterward bubbles-induced cavitation substan-
adiofrequency (RF) as a minimally invasive tool has been widely applied in different types of tumors,1−3 but the inevitable high output power and long irradiation time usually cause damages to normal tissues in clinical application.4 RF therapeutic enhancement agents (TEAs) will be an ideal solution through improving ablated volume to reduce power and time. Although magnetic metal nanoparticles on which current reports focused could heat an oscillating magnetic field component of RF field for promotion of RF ablation,5−7 high output power is still needed to obtain high oscillating magnetic field.3,7 Additionally, the toxicity and degradability of metalbased particles remain an unresolved and disputed issue. On the whole, research on RF TEAs capable of solving above-mentioned drawbacks is still challenging but urgently needed. Bubbles-induced cavitation has been widely applied in contrast-intensified ultrasound imaging, drug or gene delivery and sonodynamic therapy.8−10 Currently, it lays the foundation © XXXX American Chemical Society
Received: November 26, 2015 Accepted: January 22, 2016
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DOI: 10.1021/acsnano.5b07486 ACS Nano XXXX, XXX, XXX−XXX
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ACS Nano tially enhanced ultrasound imaging and treatment.15,16 However, these liquid perfluorocarbon-encapsulated theranostic agents featured of the transient and violent phase-transformation process and accordingly instant cavitation are inappropriate for enhancing RF ablation,17 since the special progressive temperature elevation during RF irradiation determines that the encapsulated medium of a continuous cavitation property is desirable. Moreover, the two dominant means, acoustic droplet vaporization (ADV) and optical droplet vaporization (ODV), that can induce this specific bubbles-induced cavitation process (Figure S1),18−20 are restricted by their intrinsic drawbacks, e.g., ultrasound is easily and substantially scattered by gas or bone and the penetration-depth of laser is very shallow. To overcome the inappropriate applicability of conventional microbubbles, liquid perfluorocarbon-encapsulated theranostic agents and metal-based nanoparticles in enhancing RF ablation, hereby, we introduced a new strategy, i.e., continuous cavitation principle, to continuously enhance RF ablation and lower RF power output and irradiation time. To instantiate this concept, we use biocompatible poly lactide-glycolide acid (designated as PLGA) as the platform for encapsulation and delivery of biocompatible DL-menthol (DLM) (melting point: 32−36 °C), constructing a novel PLGA-based nanocapsule, since the solid DLM displayed a distinctively continuous solid−liquid−gas (SLG) triphase transformation property that is completely applicable to RF ablation.21 Accordingly, to solve the existing issues of ADV and ODV, we herein establish a new radiofrequency solidoid vaporization (designated as RSV) process to trigger the continuous cavitation for RF-responsive theranostics of cancers. This RSV process can employ external RF-mediated heat energy to induce vaporization of encapsulated solid DLM (Figure S1), and continuously generate DLM bubbles (Figure 1A).Therefore, the PLGA-based nanocapsules can continuously enhance ultrasound contrast imaging and, furthermore, enhance RF ablation via the distinctively continuous cavitation. Importantly, systematic in vitro and in vivo investigations have been carried out to successfully demonstrate the feasibility and efficiency of RSV for cancer theranostics.
Figure 1. Principle of RSV process and structural characterization of solid DLM-encapsulated PLGA nanocapsules. (A) Schematic illustration of RSV process mediated by DLM@PLGA. (B and D) TEM images of DLM@PLGA (B) and PLGA (D), and DLM@PLGA capsules were indicated by red arrows in (B). (C and E) Confocal microscopic images of DLM@PLGA (C) and PLGA (E) after RF irradiation. Notes: the output power of RF is 3 W, and the irradiation time is 1 min; the occurrence of expansion and melting in image B results from high temperature and encapsulated DLM’s evaporation when exposure to electron beam.
in DLM@PLGA and the occurrence of RSV process. Both optical microscopic image (Figures 2b and S4) and bright field image (Figure 2d) also demonstrate such DLM@PLGA nanocapsules can respond to RF irradiations to generate DLM bubbles via this special RSV process. In Vitro and in Vivo Evaluations on Using RSV Process To Trigger RSV Process for Enhancing Ultrasound Imaging. The in vitro RF-thermal transition efficiency was first investigated to determine the following experimental parameters. A continuous RF heating behavior (Figure S5A) could be directly observed using egg albumen colloidal solution as in vivo model environment, wherein a power-dependent temperature increase profile is also obtained. It is worth noting that after adding DLM@PLGA, larger incremental amplitude of temperature is observed (Figure S5B). This phenomenon can be attributed to the DLM@PLGA nanocapsules that can incipiently reduce free diffusion-induced heat loss via bubbles’ formationmediated energy storage, and afterward release energy via bubbles’ cavitation effect, substantially improving the utilization efficiency of heat. Similar to the HIFU-triggered solidoid vaporization,21 the RSV process in this report can enhance ultrasound imaging, in other words, ultrasound imaging technology can be employed as another means to monitor the occurrence of RSV process. We found that there is no obvious change in contrast and average gray values for either PBS or PLGA suspension before and after exposure to RF field (corresponding upper and middle panels of Figure 3A,B). Comparatively, the more evident contrast-
RESULTS AND DISCUSSION Construction of Solid DLM-Encapsulated PLGA Nanocapsules (DLM@PLGA). DLM@PLGA nanocapsules can be obtained via a simple, versatile one-pot microemulsion (W/O) method, wherein hydrophobic DLM and methylene chloride can be regarded as oil phase, and PVA was adopted to avoid aggregation of the as-synthesized DLM@PLGA. In Figures 1B, 2a and S2, it is observed that the average particle size of DLM@ PLGA nanocapsules with a defined morphology is 450 nm. The obvious contrast difference between PLGA and DLM@PLGA indicates the successful loading of DLM (Figure 1B,D) with the loading amount of 15.05 wt % according to the thermogravimetry analysis (TGA) (Figure S3). IN comparison to free DLM (32−36 °C), an evident endothermic peak (Figure S3) at around 50 °C in the differential thermal analysis profile of DLM@PLGA indicates the elevation of DLM’s melting point after encapsulation by PLGA platform.22 Additionally, we found that a considerable number of menthol microbubbles with different particle diameters can be generated from DLM@PLGA suspension in the confocal microscopic image (Figures 1C, and 2c) upon exposure to RF heating (60 °C), while no bubbles are found in PLGA (Figure 1E) under the same RF parameters, confirming the successful loading of DLM B
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Figure 2. Structural characterizations of DLM@PLGA nanocapsules before and after RF heating. (a) SEM image of DLM@PLGA nanocapsules, and DLM@PLGA capsules were indicated by red arrows. (b−d) Optical (b), confocal (c), and bright-field confocal (d) microscopic images of DLM@PLGA nanocapsules after RF heating.
imaging. Similar to in vitro outcomes, in vivo results with directly injected liquid free DLM also demonstrate the presence of vaporized DLM bubbles (Figure S7b). Ex Vivo Intensified RF Ablation through RSV ProcessMediated Continuous Cavitation. In addition to contrastenhanced intelligent ultrasound imaging, the RSV process can be further employed to enhance RF ablation for tumors via the continuous cavitation principle that is completely different from previous principle of metal-mediated magnetic-heat transfer. First, the distinctively continuous cavitation was explored via evaluation of the inertial cavitation dose (ICD). Figure S10A shows ICD values of PBS, PLGA and DLM@PLGA with laser (wavelength, 980 nm; power density, 2 w/cm2) as noninvasive heat source instead of RF needle, since this power density can provide temperature high enough (above 50 °C, Figure S10C) to trigger SLG process. The whole schematic for the measurement apparatus can be seen in Figure S10B. It is clearly shown that DLM@PLGA harvests the largest ICD value, and even after 30 min, the ICD value still remains the same, demonstrating occurrence of the continuous cavitation in DLM@PLGA nanocapsules. Ex vivo experiments were carried out on degassed pork liver. Similar to in vitro results, DLM@PLGA achieves the largest incremental amplitude (ΔT = 49 °C) of the maximum temperature after exposure to RF irradiation (Figure 4a,b). Therefore, DLM@PLGA, as expected, achieves the largest ablation volume (Figure 4c,d), convincingly demonstrating the special RSV process enables the enhanced RF ablation. Noticeably, the much larger increment in ablation volume using DLM@PLGA than that using free DLM can be probably attributed to the uniformity of DLM@PLGA nanocapsules in
enhanced ultrasound imaging is observed in DLM@PLGA suspension, which can be attributed to the presence of RSVinduced DLM bubbles. Moreover, after RF-heating, the size distribution profile of DLM@PLGA suspension becomes widen and more intensity peaks emerge (Figure S6), suggesting the rupture of DLM@PLGA nanocapsules. Noticeably, free DLM can also demonstrate this point (Figure S7a), wherein large DLM bubbles existed in liquid DLM. Such a RF-responsive, contrastenhanced ultrasound-imaging displays a concentration-dependent relationship (Figure S8). Video S1 indicates DLM@PLGA can continuously generate DLM bubbles in an open plastic tube immersed into a tank full of PBS where RF was applied, confirming the occurrence of RSV process and continuous contrast-intensified ultrasound imaging. The performance of DLM@PLGA for RSV process was further evaluated in vivo on nude mice bearing HeLa cervical cancer xenograft. After intratumoral infusions of PBS, PLGA dispersion, free liquid DLM and DLM@PLGA dispersion, the mice were exposed to RF field to trigger the RSV process, and we found that the temperature in tumor is raised to above 50 °C after RF heating for 30 s (Figure S9), thus suggesting the RSV process is easily within reach. To avoid confusion caused by thermal coagulation-induced contrast enhancement under B fundamental imaging (BFI) mode,15,21 contrast harmonic imaging (CHI) mode was also employed, because CHI mode exclusively responds to microbubbles for ultrasonography.23,24 More significant contrast enhancement under both BFI and CHI modes (Figure 3C) using DLM@PLGA than that using PBS and PLGA indicates such a RF-mediated temperature elevation directly vaporized the encapsulated DLM, demonstrating the high in vivo RSV efficiency of DLM@PLGA for ultrasound C
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Figure 3. Evaluations of using RSV process to enhance ultrasound imaging in vitro and in vivo. (A) In vitro ultrasonic images under BFI mode; (B) corresponding average gray value acquired from panels A; (C) in vivo ultrasonic images under both BFI and CHI modes.
demonstrate the encapsulated solid DLM could respond to RF field to enhance RF ablation via the distinctive RSV-mediated continuous cavitation principle. The related macroscopic appearance of ablated tumor tissues with DLM@PLGA (Figure 5C) shows a sharply demarcated hemorrhagic volume on gross inspection. Similar to ex vivo results, the hydrophobic propertyinduced agglomeration and nonuniform distribution not only results in much smaller accumulative ablated volume than that using DLM@PLGA, but also leads to the discontinuous ablation behavior (three isolated ablation regions), as indicated by yellow arrows (Figure 5c3), which greatly lower treatment precision and controllability. To further investigate the molecular mechanism of this in vivo enhanced RF ablation, related pathological analysis of tumor after RF ablation was systematically investigated. After proliferating cell nuclear antigen (PCNA) assay and tunnel staining, the least proliferating cells (represented by brown color) but the most apoptotic cells (represented by brown color) are both observed in the group of DLM@PLGA (Figure 6A). Heat shock protein 70 (HSP 70) that is closely associated with hyperpyrexia-induced apoptosis was detected,25 and the gradual up-regulation of HSP 70 expression from PBS, ultimately until to DLM@PLGA (Figure 6B) indicates RSV process promotes RF
aqueous environment which is much better than that in free DLM, since DLM is hydrophobic and prone to agglomeration into blocks and nonuniform distribution (Figure S11). In Vivo Intensified RF Ablation Based on the Continuous Cavitation Principle. The mechanism of RSV process for enhancing in vivo RF ablation can be found in Figure 5A. After either intratumorally or intravenously administering DLM@PLGA, large amounts of DLM bubbles emerge via SLG triphase transformation after RF heating, achieving energy storage of RF energy and preventing diffusion-induced heat loss by blood circulation. Afterward, the emerging bubbles will explode to release energy in the form of cavitation-associated local hyperpyrexia, shock waves, microjets, etc., eventually destroying, ablating and even removing the tumor tissues. This process greatly improves the utilization efficiency of RF energy. Therefore, DLM@PLGA nanocapsules hold great potential in occluding blood vessels in cancerous tissues, thus diminishing the blood supply to the tumor. Figure 5B,E shows the tumor treated with DLM@PLGA nanocapsules in an intratumoral injection manner receives the largest incremental amplitudes in temperature and ultrasonic contrast after RF ablation, promising DLM@PLGA will obtain the largest ablated volume (Figure 5C,D). These results further D
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Figure 4. Ex vivo evaluations of using RSV process to enhance RF ablation. (a) Thermal infrared images of ex vivo pork liver after injecting PBS, PLGA and DLM@PLGA and exposure to RF irradiations at 0 s (left column) and 40 s (right column). (b) The variation profiles of maximum temperatures in (a) after injecting PBS, PLGA, free DLM and DLM@PLGA and subsequent exposure to RF irradiation. (c) Digital photos of a slice of pork liver with maximum ablation cross-sectional area using PBS, PLGA, free DLM and DLM@PLGA after RF irradiation (1 W) for 30 s. (d) Corresponding ablated volumes calculated from (c). Notes: ** represents P-value < 0.005.
Figure 5. In vivo evaluations of using RSV process to enhance RF ablation for xenografted HeLa tumor implanted in nude mice in an intratumoral injection manner. (A) Schematic illustration of in vivo RSV process for imaging-guided RF ablation for cancer. (B) Thermal infrared images of xenografted HeLa tumor after injecting PBS (b1, b2), PLGA (b3, b4),free DLM (b5, b6), and DLM@PLGA (b7, b8) and subsequent pre- (left column) and post-RF irradiation (right column). (C) Digital photos of HeLa tumor slices with maximum ablation cross-sectional area using PBS (c1), PLGA (c2), free DLM (c3), and DLM@PLGA (c4). (D) Corresponding ablated volumes calculated from panels C. (E) Doppler ultrasonic images of HeLa tumors before and after RF ablation using different samples.
ablation through an enhanced DNA damaged-mediated pathway. More importantly, only one boundary between ablation region and unablated region in either PCNA assay or tunnel staining is
clearly observed using DLM@PLGA, suggesting cavitation can be also employed to improve the ablation precision and biosafety, which is consistent with previous report.17 On the contrary, the presence of multiple isolated ablation regions leads to multiple E
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Figure 6. Pathological and biochemical analysis of ablated tumor tissues by RF. (A) Optical microscopic images of PCNA and TUNEL immunohistochemical staining of tumor slices, wherein ablated and unablated regions can be differentiated with brownish red curves, and the scale bar is 100 μm. (B) Western blot data of tumor slices, and b1−b4 represent PBS, PLGA, free DLM, and DLM@PLGA, respectively. (C) Optical microscopic images of ablated HeLa tumor slices using free DLM (c1, c2) and DLM@PLGA (c3, c4), wherein the scale bars in c1 and c3 are 100 μm and those in c2 and c4 are 50 μm. (D) Schematic image of tumor ablated by RF irradiation after injecting free liquid DLM (d1) and DLM@ PLGA nanocapsules dispersion (d2).
Evaluations on Prohibitory Growth of HeLa Solid Tumor Using Such DLM@PLGA Capsules after RF Ablation. Moreover, the therapeutic outcome using DLM@ PLGA nanocapsules in an intravenous manner was explored. It is reported that nanoparticles of around 600 nm in diameter can enter HeLa tumor through enhanced permeability and retention (EPR).26−28 More significantly, both Doppler blood ultrasonagraphy and immunohistochemical staining by CD31 and CD34 demonstrate the presence of rich blood vessels around the tumor (Figures 5E and S12), which is beneficial for delivering such PLGA-based capsules. Noticeably, such nanocapsules share an excellent structural stability in simulated body fluid (SBF) solution within 12 h under normal physiological temperature (37 °C), as shown in Figure S13, wherein no rupture and no 1,1′dioctadecyl-3,3,3′,3′-tetramethy-lindocarbocyanine perchlorate
isolated boundaries in either PCNA or tunnel staining, further demonstrating the poor precision and controllability using free DLM. Similar phenomenon can be seen in hematoxylin and eosin (H&E) staining (Figure 6C), wherein the disappearance of cell structure, fragmentation of lysed cell membranes and ruptured nuclei can be seen. This schematic explanation and depiction can be found in Figure 6D, wherein the blocks and nonuniform distribution of free DLM in tumor tissue lead to the isolated ablation region, while the ablation region enhanced by each DLM@PLGA nanocapsule is overlapped and interconnected with each other to construct an integrated continuous ablation region. Additionally, the coagulated necrosis was also accompanied by the disappearance of cell structure, fragmentation of lysed cell membranes and ruptured nuclei that were the typical features associated with necrosis. F
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Figure 7. In vivo evaluations of using RSV process to enhance RF ablation and inhibit the growth of xenografted HeLa tumor implanted in nude mice in an intravenous injection manner. (a and b) Digital photos and corresponding ablation values of HeLa tumor with maximum ablation cross-sectional area after treatments with different samples and subsequent RF ablation. (c and d) ARFI images (d) and quantitative shear wave velocities (c) of HeLa tumor treated with different samples before and after RF ablation. Notes: ** represents P-value of
