In Vivo Targeted, Responsive and Synergistic Cancer

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Biological and Medical Applications of Materials and Interfaces

In Vivo Targeted, Responsive and Synergistic Cancer Nanotheranostics by MRI-Guided Synergistic HIFU Ablation and Chemotherapy Hailin Tang, Yuan Guo, Li Peng, Hui Fang, Zhigang Wang, Yuanyi Zheng, Haitao Ran, and Yu Chen ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b01967 • Publication Date (Web): 13 Apr 2018 Downloaded from http://pubs.acs.org on April 13, 2018

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ACS Applied Materials & Interfaces

In Vivo Targeted, Responsive and Synergistic Cancer Nanotheranostics by MRI-Guided Synergistic HIFU Ablation and Chemotherapy Hailin Tang1¶, Yuan Guo2¶, Li Peng 1, Hui Fang1, Zhigang Wang2, Yuanyi Zheng4, Haitao Ran2* and Yu Chen3* 1

Department of Ultrasound, Tongde Hospital of Zhejiang Province, Hangzhou, 310012, P. R. China

2

Second Affiliated Hospital of Chongqing Medical University & Chongqing Key Laboratory of Ultrasound Molecular Imaging, Chongqing, 400010, P. R. China. Email: [email protected]

3

State Key Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai, 200050, P. R. China. Email: [email protected]

4

Shanghai Institute of Ultrasound in Medicine Shanghai Jiaotong University Affiliated Shanghai Sixth People’s Hospital Shanghai 200233, P. R. China ¶

Hailin Tang and Yuan Guo are co-first authors who contributed equally to this study.

1

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ABSTRACT As one of the most representative non-invasive therapeutic modalities, high-intensity focused ultrasound (HIFU) has shown the great promise for cancer therapy, but its low therapeutic efficacy and biosafety significantly hinder the further extensive clinical translation and application. In this work, we report on the construction of a multifunctional theranostic nanoplatform to synergistically enhance the HIFU-therapeutic efficacy based on nanomedicine. A targeted and temperature-responsive theranostic nanoplatform (PFH/DOX@PLGA/Fe3O4-FA) has been designed and fabricated for efficient ultrasound/magnetic resonance dual-modality imaging-guided HIFU/chemo synergistic therapy. Especially, the folate was conjugated onto the surface of nanoplatform for achieving actively targeting to hepatoma cells by receptor-ligand interaction, which facilitates accumulation of the nanoplatforms into tumor site. The integrated superparamagnetic iron oxide nanoparticles could generate the contrast enhancement in T2-weighted magnetic resonance imaging. By virtue of the thermal effect as generated by HIFU, liquid-gas phase transition of perfluorohexane (PFH) in nanocomposites was induced to generate PFH microbubbles, which has achieved the contrast-enhanced ultrasound imaging and significantly improved HIFU-ablation efficacy. The loaded anticancer drugs could be released from the nanocomposites at controllable manner (both pH and HIFU responsiveness). These multifunctional nanocomposites have been demonstrated to efficiently suppress the tumor growth based on the enhanced and synergistic chemotherapy and HIFU ablation, providing an efficient theranostic nanoplatform for cancer treatment. KEYWORDS: HIFU, Chemotherapy, PLGA, Nanomedicine, Synergistic therapy, Cancer

2


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1. INTRODUCTION High-intensity focused ultrasound (HIFU) is generally regarded as one of the most representative non-invasive ablation technologies that focuses high-intensity ultrasound (US) energy to ablate and destroy the tumor tissue.1-4 Especially, HIFU has been applied for the treatment of primary solid tumors and even metastatic cancer in a clinical setting.5-7 However, HIFU-based non-invasive therapy is still not satisfactory mainly because of its relatively low therapeutic efficacy in deep-seated and larger volume tumor tissue, which is typically attributed to the rapid energy attenuation along the ultrasound pathway.8-10 It often requires higher ultrasound power and longer operation duration. Currently, the low therapeutic efficacy is becoming a serious challenge for its further extensive clinical application. Some investigations have confirmed that microbubbles(MBs) can efficiently enhance the HIFU ablation outcome, because these MBs can change the acoustic environment of tissues, increase the ultrasonic cavitation.11-13 However, these lipid MBs suffer from instability, and they are easily broken with short blood circulation time. Especially, the size of traditional lipid MBs is usually too large to go through the intervals of vascular endothelial cells to tumor cells. The low stability and large dimension of commercial MBs make them difficult for continuous synergistic enhancement towards HIFU-ablation efficacy. On this ground, various nanosystems such as inorganic mesoporous silicon nanoparticles (NPs), magnetic NPs, and organic poly(lactic-co-glycolic acid) (PLGA) NPs have been constructed, which have exert the specific functionality as either ultrasound-based contrast agents or ultrasound-based synergistic agents (SAs) for tumor diagnostic imaging and therapy.14-22 Compared with large particle-sized MBs, these NPs show smaller size, longer blood-circulation time, enhanced stability and easy surface modification/engineering. However, when the size of MBs is reduced into nanoscale, the ultrasound responsiveness is unfortunately decreased significantly. Recently developed “small-to-big” strategy could efficiently solve this critical issue by designing phase-transformable small NPs.9, 17, 23-26 Based on the small particle size of NPs, they can efficiently escape from the vascular circulation and penetrate into tumor tissue, which can be further in-situ transformed into MBs with large particle size by external US triggering. As a typical paradigm, perfluorohexane (PFH) with boiling point (56℃) was 3



encapsulated into either PLGA NPs, mesoporous silica NPs or hollow mesoporous Prussian blue nanoparticles (HMPBs), which were further developed as the SAs for enhancing the HIFU-ablation efficacy.17, 27-29 These SAs could be in situ transformed into large MBs because of the temperature-responsive liquid-gas phase transition of PFH by HIFU triggering. The ultrasonic cavitation effect was increased by changing the acoustic environment within the tumor thereby boosting ultrasound energy deposition and enhancing the HIFU-ablation efficacy. In addition, HIFU ablation may unavoidably cause the existence of residual tumor cells. These residual tumor cells, however, are the culprits causing tumor recurrence and metastasis after HIFU therapy. Meanwhile, after HIFU ablation, chemotherapy drugs hardly arrive at tumor cells because most of vessels around the tumor have been damaged. Therefore, the SAs require the encapsulation of chemotherapeutic drugs and arrive around tumor cells before HIFU ablation. Chemotherapy drugs encapsulated in the SAs can deposit easily around the residual tumor cells by most of vessels of the tumor tissue damaged after HIFU ablation. Thus, these combined effects could maximally kill residual tumor cells, reduce tumor recurrence, and also decrease the side effects of chemotherapeutic drugs simultaneously. Especially, theoretically concentration of the SAs in the tumor tissue is related to therapeutic efficiency of HIFU ablation. Increasing SAs concentration in the tumor tissue within a certain range can improve the therapeutic efficacy of HIFU ablation. Considering the toxic side effects of chemotherapeutic drugs and increasing concentration of the SAs, it is highly desirable to design the SAs with efficient targeting capability. In this work, we have successfully constructed a multifunctional nanoplatform (SAs) for targeted and synergistic HIFU ablation and chemotherapy on combating tumor. The as-designed SAs consist of five individual parts. First, PLGA, as a FDA-approved agent, was selected as the nanosized matrix based on its high biocompatibility and biodegradability. Second, the folate (FA) was covalently bounded onto the outer shell of nanoparticles for targeting transportation and accumulation into tumor tissue. Third, liquid-gas phase-changeable PFH was encapsulated into the core of PLGA nanoparticles, which could be vaporized by the thermal effect of HIFU. Fourth, superparamagnetic Fe3O4 NPs were integrated into the shell of PLGA nanoparticles for endowing the nanocomposites with T2-weighted magnetic resonance (MR) imaging capability. 4


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Fifth, doxorubicin (DOX), as a typical anticancer drug, was loaded into the nanocomposites. Such a nanotheranostic agent is based on PLGA NPs with temperature-responsive PFH in the core, superparamagnetic Fe3O4 NPs in the shell, anticancer drug DOX encapsulated inside and FA decorated on the outer surface (designated as PFH/DOX@PLGA/Fe3O4-FA nanocomposites). This novel multifunctional nanotheranostic agent can serve as a promising candidate for further studies

on

cancer

therapy,

due

to

its

favorable

features

of

efficient

targeting,

temperature-responsive phase transition, responsive drug delivery and synergistic therapeutic efficiency of HIFU ablation/chemotherapy under the US/MRI imaging-guidance, which has not been achieved in previous work. The designed strategy could result in much enhanced HIFU theranostic efficacy and the biosafety of HIFU ablation, with simultaneously minimized systemic toxicity.

2. MATERIALS AND METHODS 2.1 Synthesis of PFH/DOX@PLGA/Fe3O4-FA nanocomposites The non-targeting PLGA NPs with the integrated Fe3O4, PFH and DOX were prepared by a typical double-emulsion method. In a typical process, 50 µL of oleic-modified Fe3O4 NPs (28 mg/mL) and 100 µL PFH were added into 3 mL of CH3Cl after dissolving 50 mg of PLGA crystals and 5 mg of DOX. In the first emulsion, the mixture was emulsified in an ice bath by using an ultrasonic probe with power of 133 W for 30 s. Then, the aforementioned solution was poured into 10 mL of PVA solution (5% w/v) and emulsified at the same condition for the second emulsion. The final solution was added to a 20 mL of isopropanol and placed in an ice bath overnight to stabilize the NPs and extract CH3Cl. Subsequently, the NPs were centrifuged at 9000 rpm for 10 min to remove impurities and washed with deionized water. The above process of

centrifugation

and

washing

were

repeated

for

three

times.

The

prepared

PFH/DOX@PLGA/Fe3O4 nanocomposites were stored at 4 ℃ for further use. The folate-linked PLGA targeting nanocomposites were fabricated by a carbodiimide reaction. At first, the non-targeting PFH/DOX@PLGA/Fe3O4 nanocomposites were resuspended in a MES buffer (0.1 mol/L, pH = 5.5), which was then added with coupling agents EDC (molar 5



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ratio of PLGA and EDC was 1:10) and NHS (mass ratio of EDC and NHS was 1:3) in an ice bath for 30 min with gentle stirring to activate PLGA NPs. The unreacted EDC and NHS were removed by centrifugation and washing of above NPs solution. Then, the activated NPs were incubated with MES (0.1 mol/L, pH = 8.0) buffer at room temperature for 30 min, which was dissolved with Fol-PEG-NH2. The resulting folate-NPs bioconjugates were centrifuged and washed

with

MES

buffer

for

three

times

to

remove

unbound

Fol-PEG-NH2.

PFH@PLGA/Fe3O4-FA, DOX@PLGA/Fe3O4-FA, fluorescently labeled NPs and blank PLGA NPs were fabricated by the same above procedures. 2.2 Characterization of PFH/DOX@PLGA/Fe3O4-FA nanocomposites The average particle size and Zeta potential of the PFH/DOX@PLGA/Fe3O4-FA nanocomposites were determined by a dynamic laser light scattering system (Malvern Instruments, UK).

The structure and morphological of PFH/DOX@PLGA/Fe3O4-FA

nanocomposites were characterized by a transmission electron microscope (TEM, Hitachi H-7600, Japan). 2.3 Cell culture and animal model Bel-7402 human hepatoma cells expressing folate receptors were obtained from BeNa Culture Collection (Beijing, China) and cultured in the routine way using RPMI-1640 medium with 10% fetal bovine serum and 1% penicillin/streptomycin at 37℃. All animal experiments were carried out according to the guidelines of Animal Ethics Committee of Chongqing Medical University. A certain amount of female nude mice (4-6 weeks old; weight of 16-19 g) were purchased from and fed at Animal Experiment Center of Chongqing Medical University (Chongqing, China). To inoculate solid tumor, Bel-7402 human hepatoma cells were detached with trypsin and resuspended in sterile PBS (pH = 7.4). Then, the cells (1 × 106 cells/200 µL) were subcutaneously injected into the left flank of every mouse. All tumor-bearing mice were used when the volumes reached around 200 mm3. 2.4 Evaluation of DOX-loaded PFH/DOX@PLGA/Fe3O4-FA nanocomposites 6


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To determine the DOX-loading amount and efficiency, the PFH/DOX@PLGA/Fe3O4-FA was measured by UV-VIS-NIR spectrophotometer (UV-2550 Shimadzu, Japan) and high-phase liquid chromatography (HPLC). The DOX-loading amount and encapsulation efficiency were calculated by the following equations: Loading amount (%) = weight of DOX in NPs/total weight of NPs × 100% Encapsulation efficiency (%) = weight of DOX in NPs/total weight of DOX × 100%. 2.5 The DOX-releasing behavior as triggered by HIFU and different pH values 2.5.1 PH/HIFU-triggered DOX release from PFH/DOX@PLGA/Fe3O4-FA nanocomposites To assess the DOX-releasing behavior, the PFH/DOX@PLGA/Fe3O4-FA nanocomposites in different pH conditions were initially assessed. The same volume of nanocomposite solutions in 3 mL PBS at two different pH (5.5 and 7.4) were placed in two dialysis bags (8-12 kDa MWCO) and shaken at 120 rpm at 37℃. To further analyze the DOX-releasing behavior of nanocomposites as triggered by HIFU, two samples of nanocomposites were prepared by aforementioned method. After 5 h releasing, the samples in pH 5.5 and pH 7.4 were exposed to HIFU irradiation (120 W, 5 s) (JC 200, Chongqing Haifu Technology, Chongqing, China) respectively, which was followed by shaking for another 40 h. At pre-determined time points, 1 mL of dialysis solution from each sample was collected and the same volume of fresh PBS was added back. The DOX concentration of each sample was detected by using the HPLC. 2.5.2 Intracellular DOX release promoted by HIFU The intracellular DOX-releasing behavior with or without HIFU exposure at a certain pH value was investigated. The Bel-7402 cells in logarithmic phase seeded in laser confocal culture dishes at a certain density were divided into targeting group and non-targeting group, and each of group was subdivided into two groups with or without HIFU irradiation. After 24 h incubation, different groups were received different treatments. 100 µL of PFH/DOX@PLGA/Fe3O4-FA (1 mg/mL) was added to two dishes as targeting groups and the cells were cultured for another 2 h. Subsequently, one of dishes in targeting group was triggered by HIFU using 120 W for 5 s (JC 200, Chongqing Medical University, China) and the cells were cultured for another 4 h. 100 µL 7



of PFH/DOX@PLGA/Fe3O4 (1 mg/mL) were added to the other two dishes as non-targeting groups and the cells in one of dishes received the same treatment as above. Finally, the cells in all four groups were gently washed with PBS for three times, fixed with 4% paraformaldehyde for 15 min and stained with DAPI for 10 min. Finally, the cells were washed with PBS twice and analyzed using an confocal laser scanning microscopy (CLSM) (Nikon A1R, Japan). 2.6 Biocompatibility evaluation of PFH@PLGA/Fe3O4-FA nanocomposites 2.6.1 In vitro cytotoxicity assay The cytotoxicity of PFH@PLGA/Fe3O4-FA nanocomposites to different cells were measured by the typical CCK-8 assay. Bel-7402 cells, SKOV-3 cells, MB-231 cells and hepatocyte were seeded in the 96-well plates (5 × 104 cells/well) for 12 h. Next, the fresh medium containing different concentrations of NPs (0.5, 1.0, 2.0, 4.0, 8.0 mg/mL) were employed to replace the previous medium. The wells with cells but without NPs and the wells without cells in each plate were used as control groups. After 24 h incubation, the medium was carefully removed and replaced by 10 µL of CCK-8 solution, and then the cells were incubated for another 3 h. Finally, the absorbance of each well was measured by a microplate reader (BIO-TEK EL × 800, USA) at 490 nm. Cell viability (%) was calculated by the following equation: Cell viability = (ODnanoparticles – ODblank)/(ODwithout nanoparticles – ODblank) × 100%. 2.6.2 In vivo toxicity evaluation Healthy female nude mice approximately weighted 18 g were divided into two groups randomly (n = 3 in each group). The mice in treated group were intravenously injected with a single dose of PFH@PLGA/Fe3O4-FA nanocomposites (200 µL, 5 mg/mL) and mice in another group without any treatment were used as blank control. After 21 days, all of mice were sacrificed to collect the major organs including heart, liver, spleen, lung and kidney. The organ slices were stained with hematoxylineosin (H&E) after fixed with 4% paraformaldehyde for 24 h and observed on optical microscope. 2.7 In vitro and in vivo targeting capability of PFH@PLGA/Fe3O4-FA nanocomposites 2.7.1 In vitro cell targeting 8


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Bel-7402 cells were seeded in laser confocal culture dishes at an appropriate density for 24 h and formed a monolayer at the bottom of each dish. RPMI-1640 medium was changed with fresh medium containing 100 µL of DiI-labeled targeting PFH/DOX@PLGA/Fe3O4-FA (1 mg/mL) and 100 µL of DiI-labeled non-targeting PFH/DOX@PLGA/Fe3O4 (1 mg/mL). After 2 h incubation, the cells in two groups were gently washed with PBS three times, fixed with 4% paraformaldehyde for 15 min and stained with DAPI for 10 min followed by washing with PBS. Finally, cell-targeting behavior of nanocomposites in targeting group and non-targeting group were observed by CLSM, respectively. In addition, normal hepatocyte was used and co-incubated with 100 µL of DiI-labeled targeting PFH/DOX@PLGA/Fe3O4-FA (1 mg/mL) for control. 2.7.2 In vivo tumor targeting The Bel-7402 tumor-bearing nude mice were randomly divided into targeting group and non-targeting group (n = 3 in each group) when the volumes of tumors reached to about 150 mm3. All the mice were anesthetized by isoflurane (4 L/min of oxygen flow rate, 4% anesthetic concentration, 2% maintenance concentration). In the two groups, the mice were intravenously injected with 200 µL of DiR-labeled targeting PFH/DOX@PLGA/Fe3O4-FA (5 mg/mL) and 200 µL DiR-labeled non-targeting PFH/DOX@PLGA/Fe3O4 (5 mg/mL) via the tail vein, respectively. The fluorescence signals of nanocomposites in mice were taken at 1 h, 6 h and 12 h after injection by using an FLI system (CRI Wobrun, USA). Finally, the mice were sacrificed 24 h post-injection, and the major organs (heart, liver, spleen, lung and kidney) and tumor were collected for fluorescent imaging. 2.8 In vitro and in vivo targeted ultrasound imaging after phase change of PFH in nanocomposites 2.8.1 Phase-transition performance of PFH in nanocomposites A drop of PFH/DOX@PLGA/Fe3O4-FA (0.1 mg/mL) was put on the slide, covered by a cover slip and heated with a heating panel. The phase change of the nanocomposites was visualized under an optical microscope, and images were taken before heating and at 30 s, 60 s, 120 s and 300 s after heating, respectively. In addition, a certain amount of DiI-labeled 9



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PFH/DOX@PLGA/Fe3O4-FA nanocomposites were added to a 2 mL Eppendorf tube, and the vaporization of the fluorescent nanocomposites was observed using an inverted fluorescence microscope (Olympus IX53, Canada) before and after HIFU exposure (120 W, 5 s). Simultaneously, the temperature during vaporization and time of phase change were recorded. 2.8.2 In vitro and in vivo ultrasound imaging Firstly,

the

ultrasound-imaging

performance

of

PFH/DOX@PLGA/Fe3O4-FA

nanocomposites in vitro as trigged by temperature and HIFU was evaluated, respectively. The nanocomposites were placed in 2 mL Eppendorf tube and heated in a thermostatic water bath, then transferred to 3% agarose gel (w/v) phantom according to the previous phase-transition time and immediately taken US images under B and contrast mode by using MyLab 90 (Esaote, Italy). In addition, the US imaging for the samples including PFH/DOX@PLGA/Fe3O4-FA, PFH, DOX@PLGA/Fe3O4-FA and saline control under both B mode and contrast mode was conducted as well before and after HIFU exposure (120 W, 5 s). Subsequently, the corresponding average gray values within the region of interest (ROI) were quantitatively measured by software of DFY (independently invented by the Institution of Ultrasound Imaging of Chongqing Medical University, China). Next, when volume of tumors on nude mice reached to about 150 mm3, the tumor-bearing nude mice were used to evaluate the US contrast imaging effect of PFH/DOX@PLGA/Fe3O4-FA nanocomposites. The mice were divided into targeting group and non-targeting group, anesthetized and injected via tail vein with 200 µL PFH/DOX@PLGA/Fe3O4-FA (5 mg/mL) and 200 µL PFH/DOX@PLGA/Fe3O4 (5 mg/mL), respectively. After 6 h of injection (according to the previous fluorescence imaging in vivo), US images of the tumor tissue in each group were taken before and after HIFU exposure (120 W, 5 s), and the average gray value of each tumor was detected simultaneously. 2.9 In vitro and in vivo targeted MRI imaging MR imaging performance of PFH/DOX@PLGA/Fe3O4-FA nanocomposites in vitro and in vivo were conducted on a MRI equipment (Philips Achieva 3.0 T MRI scanner, Philips Healthcare, MA). The PFH/DOX@PLGA/Fe3O4-FA at different concentration of Fe3O4 (0, 87.5, 10


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175, 350, 700, 1400 µg/mL) and pure PLGA NPs placed in 2 mL Eppendorf tubes were imaged, respectively. T2-weighted images of all samples were set using the following parameter: repetition time (TR) = 30 ms, echo time (TE) = 9.2 ms, slice thickness = 3 mm, FOV = 160 mm × 160 mm. The ROI T2 signal intensity of each sample was further measured. The nude mice bearing around 150 mm3 volume of tumor were injected via tail vein with PFH/DOX@PLGA/Fe3O4-FA

nanocomposites

(200

µL,

5

mg/mL)

and

PFH/DOX@PLGA/Fe3O4 NPs (200 µL, 5 mg/mL) as targeting group and non-targeting group, respectively. The nude mice without any treatment were used as blank control. Then, the in vivo T2-weighted images were acquired before injection and at 1 h, 6 h and 24 h after injection. The T2-weighted imaging parameters for turbo spin echo (TSE) sequences were set as follows: TR = 3200 ms, TE = 80 ms, slice thickness = 3 mm, FOV = 160 mm × 160 mm, and the T2 signal intensity of each tumor was measured. 2.10 Evaluation of synergistic effect of PFH/DOX@PLGA/Fe3O4-FA nanocomposites for targeted HIFU ablation and chemotherapy 2.10.1. Ex vivo HIFU ablation efficacy HIFU ablation effect of PFH/DOX@PLGA/Fe3O4-FA nanocomposites was evaluation on a HIFU system, which composed of a therapeutic transducer (0.8 MHz of frequency and focal distance from 135-155 mm) inverted placed at the bottom of a degassed water tank, a real-time diagnostic transducer (3.5 MH of frequency) in the center of the therapeutic transducer and computer for automated control. In addition, the therapeutic transducer was used for ablating target tissue by emitting HIFU, and the diagnostic transducer was used for locating a target tissue before HIFU ablation and simultaneously monitoring therapeutic procedure. Fresh ex vivo bovine livers with 10 cm × 10 cm × 10 cm in size were degassed for 1 h and conducted against four groups: PFH/DOX@PLGA/Fe3O4-FA,

DOX@PLGA/Fe3O4-FA,

DOX@PLGA-FA and saline. Typically, fresh ex vivo bovine livers were placed in a tank filled with degassed water. Subsequently, 200 µL NPs at 5 mg/mL (PFH/DOX@PLGA/Fe3O4-FA, DOX@PLGA/Fe3O4-FA, DOX@PLGA-FA and saline) were directly injected into the bovine livers by a 1 mL syringe, and the injection site was simultaneously monitored by a diagnostic US 11



transducer in the HIFU system. Immediately after that, HIFU ablation at different power (120 W, 150 W and 180 W) for 5 s was conducted using a therapeutic transducer on the injection site used, and then the volume of coagulated liver tissues was calculated according to measured maximum length, width and depth (mm) by the following equation: V (mm3)= π/6 × length × width × depth. Furthermore, gray-scale changes of coagulated liver tissues in four groups before and after HIFU exposure were recorded on the diagnostic ultrasound images for monitoring synergistic HIFU effect of NPs during HIFU ablation procedure. 2.10.2. In vivo chemo/HIFU synergistic therapy effect The hepatocellular tumor-bearing nude mice were randomly divided into the following eight groups (n = 3 in each group) with different treatment: HIFU combined with PFH/DOX@PLGA/Fe3O4-FA + HIFU, HIFU combined with PFH@PLGA/Fe3O4-FA, HIFU combined with DOX@PLGA/Fe3O4-FA, HIFU combined with PFH/DOX@PLGA/Fe3O4, PFH/DOX@PLGA/Fe3O4-FA, HIFU combined with saline, free-DOX and saline. Six hours after the mice received the intravenous injection of 200 µL of PFH/DOX@PLGA/Fe3O4-FA, PFH@PLGA/Fe3O4-FA, DOX@PLGA/Fe3O4-FA, PFH/DOX@PLGA/Fe3O4 (5 mg/mL) and saline, HIFU ablation against tumor was performed. In general, the mice were placed on the degassed water tank in a supine position after anesthetized with 1% pentobarbital (8 mL/kg). The mouse together with tank was moved to an appropriate orientation by a computer control and monitored through the diagnostic US imaging before ablation. Next, the HIFU exposure was conducted by the therapeutic US transducer at a power of 120 W for 5 s under a real-time guidance of diagnostic US transducer. After aforementioned treatment, the tumor volumes of all mice were recorded with a three-days interval. In addition, all mice were sacrificed 21 days after treatment and tumors of mice were harvested for H&E staining to observe apoptosis and necrosis. 2.11 Statistical analysis All Enumeration data were expressed as mean ± standard deviation (SD). Significant differences among groups were analyzed using a one-way ANOVA and differences for individual groups were determined using Student’s t-test. The results were regarded as a significant 12


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difference when P < 0.05.

3. RESULTS AND DISCUSSION 3.1 Design, synthesis and characterization of PFH/DOX@PLGA/Fe3O4-FA nanocomposites

Scheme 1. Schematic illustration of the synthetic process for PFH/DOX@PLGA/Fe3O4-FA nanocomposites, and corresponding theranostic functionality for targeted US/MR imaging-guided HIFU/chemo synergistic cancer therapy.

In

this work,

nanocomposites

we successfully synthesized temperature-responsive and targeted

(PFH/DOX@PLGA/

Fe3O4-FA)

for

synergistic

HIFU

ablation

and

chemotherapy on combating cancer. As shown in Scheme 1, the designed nanocomposites includes PLGA, folate, Fe3O4, PFH and DOX, which were elaborately integrated together to achieve the theranostic and synergistic functionality. Especially, the PLGA matrix is highly biocompatible and biodegradable. It has been demonstrated that the hepatocellular tumor-bearing 13



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mice model, that was implanted with Bel-7402 human hepatoma cells, expresses folate receptors. Initiative conjugations between folate ligands decorated on the surface of NPs and folate receptors on the surface of tumor cells in situ was critical to realize the targeting efficiency of NPs, which was demonstrated in this work. The liquid PFH with desirable boiling point of 56°C was encapsulated in the core of NPs, which could change to gas phase by thermal effect of HIFU and enhance the HIFU-ablation efficacy subsequently. The superparamagnetic Fe3O4 nanoparticles was integrated into the shell of nanocomposites to act as negative contrast agents for T2-weighted MRI, and the DOX as the anticancer drug was loaded into the nanocomposites for enhanced chemotherapy.

Figure

1.

Characterization

of


nanocomposites.

TEM

image

of

PFH/DOX@PLGA/Fe3O4-FA nanocomposites at (A) low and (B) high magnifications. (C) DLS-based size distribution and (D) Zeta potential of PFH/DOX@PLGA/Fe3O4-FA nanocomposites.

After PFH/DOX@PLGA/Fe3O4-FA nanocomposites are intravenously injected into the hepatocellular tumor-bearing mice, these nanocomposites easily pass through the vessel endothelial gaps of tumor due to the initially nanoscale size (Scheme 1). Additionally, the nanocomposites can specifically target to tumor cells by means of initiative conjugations between folate ligands decorated on the surface of NPs and folate receptors expressed by tumor cells. The Fe3O4 NPs embedded in shell of nanocomposites can be utilized for precise preoperative imaging diagnosis and intraoperative imaging guidance. Moreover, Fe3O4 NPs can 14


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enhance the sensitivity of tumor to hyperthermia effect.30 By further temperature increase caused by thermal effect of HIFU, PFH in PFH/DOX@PLGA/Fe3O4-FA NPs can be transformed from liquid to gas, and such post-formed PFH MBs can be not only used to improve ultrasound imaging but also capable of increasing the therapeutic efficacy of HIFU. Furthermore, HIFU exposure and the intrinsic tumor-acidic microenvironment can accelerate the DOX release from the NPs, thereby synergizing the efficacy of HIFU/chemotherapy. The synthetic process of PFH/DOX@PLGA/Fe3O4-FA nanocomposites was based on the fabrication of the folate-modified NPs by a double emulsification method and carbodiimide technique (Scheme 1 and Figure S1). Transmission electron microscope (TEM) image of PFH/DOX@PLGA/Fe3O4-FA nanocomposites shows the well-defined spherical morphology and high dispersity (Figure 1A). In high-magnification TEM image (Figure 1B), some dark NPs inserted in the spherical shell can be directly observed, indicating that the Fe3O4 NPs are successfully integrated into PLGA shell. The average diameter and Zeta potential of PFH/DOX@PLGA/Fe3O4-FA nanocomposites are in range of 281.0 ± 79.6 nm with a polydispersity index of 0.139 (Figure 1C) and -5.49 ± 5.32 mV (Figure 1D), respectively. These characterization results indicate that the developed nanocomposites with small size and negative surface potential would accumulate into tumor tissues by a mechanism known as the enhanced permeability and retention (EPR) effect.31-35 It has been well demonstrated that, different from the vessels in healthy tissues, the vasculatures of solid tumor are more poorly differentiated, thus making nano-sized particles easily extravasate to tumor tissues and improving the circulation time of the nanosystems.36

3.2 DOX-loading and stimuli-responsive drug release as triggered by pH and/or HIFU exposure Figure 2A shows that DOX was successfully embedded into PFH/DOX@PLGA/Fe3O4-FA nanocomposites, which was verified by UV-vis spectra. The DOX encapsulation efficiency and loading amount were determined as 50.31 ± 5.12% and 5.03 ± 0.51%, respectively. The DOX-loading amount of the NPs in absence of PFH was 5.28±0.23%. In addition, as shown in 15



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Figure 2B, the DOX encapsulation efficiencies of the nanocomposites at different iron concentration (43.75, 87.50, 175.00, 350.00, 700.00, 1400.00 µg/mL) were around 51.01 ± 4.46%, 53.40 ± 1.85%, 48.13 ± 2.60%, 52.66 ± 0.47%, 54.29 ± 0.98%, 47.30 ± 4.00%, respectively, and the corresponding loading amounts were about 5.11 ± 0.44%, 5.34 ± 0.19%, 4.81 ± 0.26%, 5.27 ± 0.46%, 5.43 ± 0.98%, 4.73 ±0.40%, respectively. The aforementioned results suggest that the addition of PFH and Fe3O4 NPs into the nanocomposites has no significant influence on the encapsulation efficiency and loading amount of DOX.

Figure 2. DOX loading and stimuli-responsive drug-releasing behavior. (A) UV-vis spectra of DOX before and after encapsulation into the nanocomposites (inset: digital photos of DOX before (left image) and after loading into nanoparticles (right image)). (B) The encapsulation efficiency of DOX into the nanocomposites at different Fe3O4 concentrations. (C) The DOX-releasing patterns from PFH/DOX@PLGA/Fe3O4-FA nanocomposite at different pHs and HIFU-exposure conditions. (D) Intracellular DOX-releasing behavior of targeted

nanocomposites

(PFH/DOX@PLGA/Fe3O4-FA)

and

non-targeted

nanocomposites

(PFH/DOX@PLGA/Fe3O4) with or without HIFU exposure, respectively. (E) Quantitative analysis of intracellular DOX fluorescence intensity under different treatment conditions. 16


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Controlled drug release upon an appropriate stimulus, such as pH, ultrasound, or temperature changes, plays an important role in facilitating delivery of chemotherapeutic carriers to the desired sites while minimizing drug leak in else tissues.29, 37-42 To determine the effects of HIFU and pH on DOX release from the nanocomposites, the drug-releasing profiles of PFH/DOX@PLGA/Fe3O4-FA nanocomposites were further investigated by high performance liquid chromatography (HPLC). The DOX release from PFH/DOX@PLGA/Fe3O4-FA NPs was firstly studied at pH 5.5 and pH 7.4, respectively. As shown in Figure 2C, the cumulative release percentage of DOX at pH 7.4 was only 3.81% at 5 h, whereas the percentage substantially increased to 20.76% when the pH value was reduced to 5.5, presumably due to the higher dissolvability of DOX in relation to a lower pH.43 It is known that pH of the intracellular environment is around 5.0, which is lower than that of tumor microenvironment (pH: 6.0-6.8) and normal tissues (pH: 7.2-7.4).44-45 It means the acidity-sensitive releasing profile of PFH/DOX@PLGA/Fe3O4-FA nanocomposites can be controlled by the surrounding environment. Furthermore, it was observed that a remarkably increase was achieved in the drug release at both pH 7.4 and pH 5.5 conditions after HIFU exposure at 5 h (Figure 2C). In particular, upon HIFU exposure, over 75% of total DOX was released at pH 5.5 after 45 h, and the releasing percentage was about 4-fold greater than that at pH 7.4 (18.64%), indicating that the DOX release can be further accelerated by HIFU irradiation. The cellular uptake of the nanocomposites by Bel-7402 cells and their releases of the entrapped DOX as triggered by HIFU were further evaluated. Bel-7402 cells were incubated with

targeted


nanocomposites

and

non-targeted

PFH/DOX@PLGA/Fe3O4 NPs for 24 h, respectively. After HIFU exposure (120 W, 5 s), the cells were incubated for another 4 h. The red fluorescence of DOX in targeted group treated with HIFU irradiation was mainly in cell nuclei and significantly stronger than that without HIFU exposure. Comparatively, both in non-targeted group with or without HIFU exposure, the DOX fluorescence could hardly be observed (Figure 2D). In addition, the corresponding quantitative analysis of the DOX fluorescence intensity was measured (Figure 2E). The fluorescence intensities in targeted groups were remarkably stronger than that in non-targeted groups. It is also noteworthy that the cells treated with targeted 17



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PFH/DOX@PLGA/Fe3O4-FA nanocomposites plus HIFU exhibited the highest fluorescence intensity among all groups. The fluorescence intensities in targeted PFH/DOX@PLGA/Fe3O4-FA nanocomposites plus HIFU groups were remarkably stronger than that of targeted PFH/DOX@PLGA/Fe3O4-FA NPs without HIFU groups. These results can be attributed to three major factors. The first one is targeting function of targeted PFH/DOX@PLGA/Fe3O4-FA nanoparticles. The second reason is the thermal effect generated by HIFU, and the third one is the cavitation effect of ultrasound waves. It is believed that due to the thermal effect of HIFU, the droplet-to-bubble vaporization of PFH in nanocomposites can be triggered, which can induce temporary destabilization of NPs and increase of cell membranes permeability, thus promoting drug uptake and release.43 Consequently, these PFH/DOX@PLGA/Fe3O4-FA nanocomposites have the potential to respond to the intrinsic (low pH in tumor) and external (HIFU exposure) stimuli for controllable drug release, thereby achieving the synergistic HIFU ablation and chemotherapy with minimal side effects.

3.3 In vitro and in vivo toxicity of PFH@PLGA/Fe3O4-FA nanocomposites

Figure 3. In vitro and in vivo biosafety evaluation of PFH@PLGA/Fe3O4-FA nanocomposites. (A)Cell viabilities of Bel-7402, SKOV-3, MB-231 and hepatocyte cells after incubation of various concentrations of nanocomposites for 24 h, as determined by the typical CCK-8 assay. (B) Histopathological analysis of the major organs (heart, liver, spleen, lung and kidney) in healthy female nude mice with or without receiving the nanocomposites. These organs were stained with H&E and observed under a light microscope.

To

evaluate

the

biosafety

and

biocompatibility

of

drug-free

nanocomposites

(PFH@PLGA/Fe3O4-FA), the cell cytotoxicity and in vivo toxicity on healthy nude mice were 18


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analyzed. Cell viability result showed no significant cytotoxicity to several cell lines such as Bel-7402 cells, SKOV-3 cells, MB-231 cells and hepatocyte even the concentration of nanocomposites increased to as high as 8 mg/mL (Figure 3A). Furthermore, the histopathology analysis of major normal organs (heart, liver, spleen, lung and kidney) also exhibited no noticeable damage was observed in both treated group with PFH@PLGA/Fe3O4-FA NPs and un-treated group (Figure 3B). These biocompatibility results indicate that the as-designed nanoplatform can be a potentially safe synergistic agent for biomedical imaging and multi-modality therapy of cancer.

3.4 In vitro and in vivo targeting efficacy of PFH/DOX@PLGA/Fe3O4-FA nanocomposites

Figure 4. In vitro and in vivo targeting behaviors of PFH/DOX@PLGA/Fe3O4-FA nanocomposites. (A)The binding efficiency between folate and the nanocomposites as observed by CLSM and corresponding quantitative analysis by flow cytometry. (B) CLSM images and the corresponding quantitative analyses of Bel-7402 cells after incubation with targeted or non-targeted nanoparticles. Hepatocytes incubated with targeted nanoparticles were set as the control (Green: DiO-labeled cell membrane, red: DiI-labeled nanocomposites). (C) In vivo targeting efficiency before of the PFH/DOX@PLGA/Fe3O4-FA nanocomposites after their intravenous administration. FIL images of tumor-bearing nude mouse at pre-injection and 1 h, 6 h, 12 h post-injection of (a-d) targeted nanocomposites and (e-h) non-targeted nanocomposites as labeled by DiR dye. (D) The corresponding in vivo quantitative fluorescence intensity of tumor. The fluorescence intensity in 19



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targeted group was significantly higher than that in non-targeted group 6 h after injection of nanocomposites (p

In Vivo Targeted, Responsive and Synergistic Cancer

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