Ablative Focused Ultrasound Synergistically Enhances Thermally

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Ablative Focused Ultrasound Synergistically Enhances Thermally Triggered Chemotherapy for Prostate Cancer in Vitro Jaspreet Singh Arora, Hakm Yousef Murad, Stephen Ashe, Gray Halliburton, Heng Yu, Jibao He, Vijay T. John, and Damir B. Khismatullin Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00216 • Publication Date (Web): 07 Jul 2016 Downloaded from http://pubs.acs.org on July 13, 2016

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Molecular Pharmaceutics

Ablative Focused Ultrasound Synergistically Enhances Thermally Triggered Chemotherapy for Prostate Cancer in Vitro Jaspreet S. Arora1,2,†, Hakm Y. Murad 3,4,†, Stephen Ashe1, Gray Halliburton3,4, Heng Yu3,4, Jibao He1, Vijay T. John1,2, and Damir B. Khismatullin3,4,5,* 1

Department of Chemical and Biomolecular Engineering, Tulane University, New Orleans, LA

2

Vector-Borne Infectious Disease Research Center, Tulane University, New Orleans, LA

3

Department of Biomedical Engineering, Tulane University, New Orleans LA

4

Tulane Institute for Integrative Engineering for Health and Medicine, Tulane University, New Orleans LA

5

Tulane Cancer Center, Tulane University School of Medicine, New Orleans, LA

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Abstract High-intensity focused ultrasound (HIFU) can locally ablate biological tissues such as tumors, i.e., induce their rapid heating and coagulative necrosis without causing damage to surrounding healthy structures. It is widely used in clinical practice for minimally invasive treatment of prostate cancer. Non-ablative, low-power HIFU was established as a promising tool for triggering the release of chemotherapeutic drugs from temperature-sensitive liposomes (TSLs). In this study, we combine ablative HIFU and thermally triggered chemotherapy to address the lack of safe and effective treatment options for elderly patients with high-risk localized prostate cancer. DU145 prostate cancer cells were exposed to chemotherapy (free and liposomal Sorafenib) and ablative HIFU, alone or in combination. Prior to cell viability assessment by trypan blue exclusion and flow cytometry, the uptake of TSLs by DU145 cells was verified by confocal microscopy and cryogenic scanning electron microscopy (cryo-SEM). The combination of TSLs encapsulating 10 µM Sorafenib and 8.7W HIFU resulted in a viability of less than 10% at 72 h post treatment, which was significant less than the viability of the cells treated with free Sorafenib (76%), Sorafenib-loaded TSLs (63%), or HIFU alone (44%). This synergy was not observed on cells treated with Sorafenib-loaded non-temperature sensitive liposomes and HIFU. According to cryo-SEM analysis, cells exposed to ablative HIFU exhibited significant mechanical disruption. Water bath immersion experiments also showed an important role of mechanical effects in the synergistic enhancement of TSL-mediated chemotherapy by ablative HIFU. This combination therapy can be an effective strategy for treatment of geriatric prostate cancer patients. Keywords: Chemotherapy; Sorafenib; Temperature-Sensitive Liposome (TSL); High-Intensity Focused Ultrasound (HIFU); Tumor Ablation; Prostate Cancer 2 ACS Paragon Plus Environment

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Introduction With 1.4 million cases diagnosed and 293,000 deaths recorded in 2013, prostate cancer became the most incident and sixth deadliest cancer in men worldwide.1 The majority of prostate cancer patients are elderly men, over the age of 65,2 who are at high risk for complications after a common, aggressive treatment such as surgery, radiation therapy, or chemotherapy.3-6 “Watchful waiting” without any curative treatment is often recommended for such high-risk patients even if their cancer is advanced7 or the patients themselves want to choose aggressive therapy.8 Meanwhile, a number of clinical studies indicate that prostate cancer patients in the watchful waiting group have poor outcomes, e.g., increased expression of metastasis markers,9 long-term distress,10 and reduced survival,11 than patients who underwent curative treatment. These data clearly show that there is a need for alternative treatment options for prostate cancer, e.g., minimally invasive ablative therapies, that can be tolerated by a geriatric population. One of the minimally invasive methods with the highest promise for prostate cancer treatment is high-intensity focused ultrasound (HIFU).12,

13

It is a radiation-free, trans-rectal,

robotically controlled therapy that uses the energy of ultrasound waves to thermally and mechanically ablate tumor cells.14,

15

HIFU is widely used as ablative anti-cancer therapy in

Canada, European and Asian countries,16,

17

and has been recently approved by the FDA for

treating prostate cancer patients in the United States.18 HIFU is effective for destruction of small localized tumors but its standalone application to the prostate gland is often accompanied by side effects such as stress incontinence, loss of potency, bladder neck and urethral stenosis.19 Standalone HIFU leads to incomplete ablation and cancer recurrence when applied to tumors greater than 3 cm in size, multifocal tumors, or tumors located in the prostate apex.20 Multiple shots of HIFU are required to cover the entire tumor volume, which dramatically increases the 3 ACS Paragon Plus Environment


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procedure time and a risk of complications due to HIFU beam misalignment.

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21, 22

This

necessitates a combination therapy that can affect large areas of the prostate with relatively low intensities and fewer shots of ablative HIFU. It should be noted that combination treatments with multiple types of therapies, e.g., radiation therapy, brachytherapy, and hormonal therapy, have proven to be an effective choice to reduce cancer recurrence.23 Chemotherapy is a last-hope treatment for patients with metastatic cancer because it can destroy cancer cells spread beyond the organ of origin. Recent studies also show that the survival rate of high-risk localized prostate cancer patients originally treated with radiation therapy and hormonal therapy increases after adjuvant chemotherapy.24 An important class of cancer drugs is tyrosine kinase inhibitors 25. One of such drugs, Sorafenib, is currently in phase 2 clinical trials for metastatic castration resistant prostate cancer.26 Sorafenib is hydrophobic and its aqueous solubility ranges from 0.00034 mg/ml at pH 1.0 to 0.00013 mg/ml at pH 4.5, which makes it practically insoluble.27 Due to the low solubility of hydrophobic chemotherapeutic drugs, large doses are necessary to destroy the entire tumor mass, leading to significant side effects of prostate cancer chemotherapy such as hot flushes, anemia, impotence, depression and gastrointestinal symptoms.28 Encapsulating chemotherapeutic drugs in carriers such as liposomes is a viable strategy to provide the required dosage which has led to several commercially available formulations, e.g., Doxil, DaunoXome and Myocet.29 Some liposomal formulations, known as temperature-sensitive liposomes (TSLs), can be thermally triggered to quickly release their contents.30-32 Ta et. al. (2014) recently demonstrated that HIFU induced heating can trigger drug release from TSLs.33 Since the absorption of the acoustic wave energy at the HIFU focus leads to elevated temperatures, HIFU can significantly increase the efficacy of thermally triggered chemotherapy 4 ACS Paragon Plus Environment

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in addition to having its own therapeutic effect. The idea to combine TSLs and focused ultrasound has been explored in a number of in vivo and in vitro tumor models.34-38 However, in these studies, focused ultrasound was non-ablative, i.e., it was used at low power to elevate the tissue temperature slightly above the physiological temperature at which TSLs can quickly release the chemotherapeutic agent. Here, we investigate the therapeutic potential for ablative HIFU combined with Sorafenib-loaded TSLs in vitro. Specifically, this work tests the hypothesis that thermal ablation and cell membrane erosion due to HIFU-induced mechanical stresses lead to the synergistic enhancement of liposomal chemotherapy for high-risk localized prostate cancer. The prostate cancer cells treated with HIFU, non-temperature sensitive liposomes (NTSLs), TSLs, a combination of HIFU and NTSLs, or a combination of HIFU and TSLs are analyzed for viability, structural damage, and liposomal uptake. Experimental Section Synthesis of liposomes The liposomes were prepared by the thin-film evaporation technique where lipids are first dissolved in an organic solvent and then evaporated to obtain a thin lipid film. The lipids 1,2DiPalmitoyl-sn-glycero-3-PhosphoCholine (DPPC, Purity > 99%) and Hydrogenated Soy L-αPhosphatidylCholine, (HSPC, Purity > 99%) (Avanti Polar Lipids, Alabaster, AL) and cholesterol (Purity > 99%, Sigma Aldrich, St. Louis, MO) were used to make liposomes. DPPC, HSPC and cholesterol at a molar ratio of 55.6:27.8:16.6 were dissolved in a solvent mixture (10ml) of chloroform and methanol (2:1 v/v), to prepare traditional TSLs with a glass transition temperature of 42-45°C.38-41 For the NTSLs, DPPC alone was dissolved in the solvent mixture. The lipid composition does not include agents such as PEGylated lipids which increase the



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circulation time of the liposomes as the TSLs are intended to be locally injected at the tumor site. To prepare drug-encapsulated liposomes, Sorafenib (Purity > 99%, Eton Bioscience, San Diego, CA) was added to the lipids and dissolved in the solvent mixture because of the hydrophobicity of the drug. The solution of the dissolved lipids with or without drug was evaporated inside a round bottom flask using a rotary evaporator R-205 (Buchi, New Castle, DE) for 2.5 hours to obtain a dry lipid film. The lipid film was then hydrated for 1 hour with 5 ml 1X Phosphate Buffer Saline (PBS, 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4) at 50ºC and 125 rpm. The hydrated solution was extruded 11 times through a 400 nm polycarbonate Whatman membrane (GE Healthcare, Aurora, OH) at 50ºC followed by the extrusion through a 100 nm Whatman membrane. To fabricate fluorescent liposomes, the lipid film was hydrated with a solution of 0.1wt% rhodamine (Purity > 95%, Sigma Aldrich, St. Louis, MO) instead of PBS. Rhodamine was encapsulated inside the aqueous core of the liposomes. It was not covalently attached to any of the lipids. The extruded solution was passed through a gel column (PD-10 Desalting Columns, GE healthcare, Piscataway, NJ) to separate the unencapsulated species (dye or drug) from the liposomes. The gel column comprised of a Sephadex G-25 medium with a particle size range of 85-260 µm. The exclusion limit of the column is 5000 Da. Since Sorafenib is not soluble in water, most of the drug was encapsulated in the bilayer during hydration and extrusion. Finally, when the liposome solution was passed through the gel column, it ensured that the entirety of Sorafenib was encapsulated in the bilayer of the liposomes. Physical characterization The liposomes were imaged by cryogenic transmission electron microscopy (Cryo-TEM) using a G2 F30 Tecnai (FEI, Hillsboro, OR) operated at 200 kV. Prior to imaging, the sample was blotted onto LC200 copper grids (Electron Microscopy Sciences, Hatfield, PA) using an 6 ACS Paragon Plus Environment

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automated system, Vitrobot (FEI, Hillsboro, OR), operated at 100% humidity. The zeta potential of liposomes was measured using a Nanobrook Omni instrument (Brookhaven, Holtzville, NY). ImageJ 1.42q (National Institutes of Health) was utilized to measure the diameter of the liposomes from their Cryo-TEM images. The image scale bars were used as a reference. A minimum of 20 liposomes were measured and their average diameter was then computed along with the standard deviation. Prostate cancer cell culture Metastatic prostatic carcinoma cell lines DU145 and PC3 (ATCC, Manassas, VA) were used to prepare the in vitro model of high-risk localized prostate cancer. Cancer cells were cultured in T-75 tissue culture flasks in a 37˚C humidified incubator with 5% CO2. The growth medium was Dulbecco’s Modified Eagle’s Medium (DMEM, ATCC, VA) supplemented with 10% fetal bovine serum, and 100 units/100 mg/mL of penicillin/streptomycin. When the cells reached 60% confluence, they were harvested for experiments, 2.7 million cells per 100 µL were trypsinized and suspended in full growth medium inside a thin-wall 0.2-mL PCR tube (Bio-Rad, Hercules, CA). The stock solutions of Sorafenib alone, Sorafenib-loaded TSLs, or Sorafenibloaded NTSLs were diluted by addition to the cell culture medium to a desired Sorafenib concentration (e.g., 10 µM). The cells were incubated with Sorafenib or Sorafenib-encapsulated liposomes for 4 hours to allow for uptake and then rinsed with PBS prior to harvesting for HIFU exposure. High-Intensity Focused Ultrasound HIFU experiments were conducted using a 1.1 MHz single element, focused ultrasound transducer (H102, Sonic Concepts, Bothell, WA) with bandwidth from 0.75 to 1.4 MHz and 7 ACS Paragon Plus Environment


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geometric focal length of 63 mm following a schematic from our previously published protocol.42 The transducer has a stainless steel housing with active diameter of 64 mm. It was placed inside a heated 37 °C chamber filled with degassed water. A 33220A function generator (Agilent Technology, Santa Clara, CA) produced an input sinusoidal signal that passed through a fixed gain (50 dB) ENL 2100L power amplifier (Electronics & Innovation, Rochester, NY) and then entered the transducer. The HIFU signal strength was monitored using a 2 Giga-samples/s InfiniiVision DSO-X-2014A oscilloscope (Agilent Technology, Santa Clara, CA). Temperature near a cancer cell pellet was measured during HIFU targeting by a mini-hypodermic Copper Constantan type T 200-µm thick bare-wired thermocouple (Omega Engineering, Stamford, CT, USA) connected to a temperature meter (SDL200, Extech Instruments, Waltham, MA). Prior to HIFU exposure, DU145 prostate cancer cells (2.7 million per 100 µL of culture medium) treated or untreated with a chemotherapeutic agent were trypsinized and centrifuged. The old growth medium containing free liposomes / free drug was discarded. And the remaining cell pellet was placed in a thin-wall 0.2 mL PCR tube. According to Liu et al,43 the inhomogeneous absorption of acoustic waves at the PCR tube wall lead to non-uniform spatial temperature fluctuations of less than 3.0 °C. The cell pellets were positioned within the HIFU focus and then exposed to HIFU at acoustic output power of 4.1 W, 8.7 W, or 12 W, which corresponds to the spatial peak temporal average intensity ISPTA of 0.38, 0.70, or 0.88 kW/cm2. No additional physical forces were used to stir the cells before or during HIFU exposure to avoid the breakup of pellets before ultrasound treatment and to ensure that all the cells remain within the focus of the ultrasound transducer and experience full exposure. After this procedure, the cells were resuspended in fresh growth medium. In all experiments, HIFU was operated in a continuous mode and its exposure time was 30 s.


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Imaging The uptake of TSLs by DU145 cells was confirmed by confocal laser microscopy (Nikon A1, Nikon Instruments, Melville, NY) and cryo-SEM (S-4800, Hitachi, Japan). Rhodamineencapsulating TSLs were incubated with the cells in a 6 well plate with glass well bottoms (MatTek, Ashland, MA) for 4 hours before confocal imaging with a 20X objective. For cryoSEM imaging, the cells were incubated with TSLs and then centrifuged to separate the free liposomes. This cell pellet was then resuspended in the culture medium and a drop of the cell suspension was plunged in liquid nitrogen to enable vitrification. The cut-section images of the cells were obtained by fracturing them at −130 °C using a flat edge knife. The samples were sublimed at −95 °C for 5 min to remove surface-vitrified water and expose surface morphology, and then sputtered with a gold−palladium composite at 11 mA for 88 s. Imaging was done at a voltage of 3 kV and a working distance of ∼8 mm. Cryo-SEM images of DU145 cells were also acquired immediately after HIFU exposure. Cell Viability Tests The viability of the cancer cells, pretreated or not with a chemotherapeutic agent, were measured post HIFU exposure by a trypan blue exclusion test and flow cytometry employing an Alexa Fluor® 488 Annexin V/Dead Cell Apoptosis Kit (ThermoFisher Scientific, Waltham, MA). After HIFU exposure, the cells from pellets were reseeded into T-25 tissue culture flasks with 5 mL of culture medium per flask and their viability was measured at 2, 24 or 72 h. In flow cytometric analysis, a typical forward- and side-scatter gate was set to exclude aggregates and particulates. A total of 100,000 events in the gate were collected using the Attune® Acoustic Focusing Cytometer (Applied Biosystems, Grand Island, NY).


The Annexin V/Dead Cell


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Apoptosis Kit stains for apoptotic cells and necrotic cells through green Annexin V and red Propidium Iodide. In the latter measurements, early apoptotic cells were identified as Propidium Iodide negative and Annexin V positive, whereas the cells identified as Propidium Iodide positive and Annexin V positive were considered late apoptotic/necrotic. To determine whether our combined treatment was additive (independent) or synergistic, the following formula for cell viability VA after additive treatment was used: VA =

VCVH , 100 %

(1)

where VC is the percentage viability after chemotherapeutic agent treatment alone and VH the percentage viability after HIFU exposure alone. The cell viability after a synergistic treatment should be significantly less than VA. Equation (1) describes the total probability of cell survival after a sequential application of two independent treatments. Please note in our experiments each part of the combined treatment was applied sequentially to the cells. Dose Dependency Sorafenib-loaded TSLs were added to the culture medium and allowed to be taken up by the DU145 cells for 4 hours. The cells were pretreated with TSLs at different concentration of Sorafenib (10 µM, 20 µM, and 50 µM) and then exposed or not exposed to 8.7W HIFU. Their viability was then measured at 2, 24, and 72 h. Water Bath v/s HIFU comparison To understand how the effects of HIFU are more than just simple heating, cell viability was measured after exposure to equivalent elevated temperatures using a water bath. A thermocouple placed within a PCR tube was immersed into a water bath heated to temperatures


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equal to those measured at HIFU powers of 4.1 W, 8.7 W, and 12 W. When a PCR tube with the cell pellet was immersed in a water bath at a particular temperature (45°C, 50°C or 60°C), temperature inside the tube was constantly measured by a thermocouple until it equilibrated to the water bath temperature. After this, the PCR tube kept in the water bath for 30 s to provide the same thermal conditions as during HIFU exposure. Cell viability in water bath vs HIFU exposure was compared using trypan blue exclusion. Statistical Analysis Three to five independent experiments per group were conducted. Statistically significant differences were set to p < 0.05 between experimental groups. The results were evaluated with ttest and ANOVA using GraphPad Prism version 5.0.2 (GraphPad Software, La Jolla, CA). The statistical data were presented as mean ± standard error of the mean (SEM). Results Physical characterization and uptake of liposomes Sorafenib-loaded TSLs are mostly unilamellar with an average diameter of 147.12 ± 39.75 nm, as seen in their cryo-TEM images (Figure 1). Their zeta potential is 0.11 ± 1.59 mV, i.e., they do not have significant surface charge. This is expected because all the lipids used in their preparation were neutral. The cryo-TEM images of Sorafenib-loaded NTSLs are in the supporting information section S1. Their average diameter is 115.83 ± 22.38 nm and their zeta potential was measured as 3.62 ± 0.98 mV. It was verified that the liposomes are stable in cell media containing 10% FBS and 50% FBS for 2.5 hours (see supporting information Figure S2).



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Figure 1. Cryo-TEM images of Sorafenib-loaded TSLs. The scale bar is 200 nm. The uptake of rhodamine-encapsulating TSLs by DU145 cells after 4 hours of incubation is shown in Figure 2. Some TSLs attached to the surface of the cells (compare Figure 2A-B and Figure 2C-D). When the cells were imaged by confocal microscopy, most TSLs were shown to be inside the cells (see red fluorescence regions in Figure 2E-F). Here, Figure 2Ei is the image of the cell where the bright field and red fluorescence signals are merged. TSLs were present everywhere in the intracellular space excluding a large region close to the cell center, which could be the cell nucleus (Figure 2Eii). The observation that TSLs do not enter the nucleus is supported by recent work by Al-Ahmady et al.40 The cell remained intact after uptake of TSLs (Figure 2Eiii). The three-dimensional distribution of rhodamine-encapsulating TSLs, obtained from confocal z-stack images, clearly show that TSLs were in the cell cytoplasm (Figure 2F). Confocal images showing the uptake of NTSLs are in the supporting information section S2. In our previously published work,44 we verified the uptake of liposomes by cancer cells where the


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Figure 2. DU145 prostate cancer cells uptake TSLs. A and B: Low and high resolution cryoSEM images of untreated cells. C and D: High resolution cryo-SEM images of the cells incubated with rhodamine-encapsulating TSLs for 4 hours. Vesicular bodies (red circles) are spotted on the cell surface. E: Confocal image of the cell incubated with rhodamineencapsulating TSLs. Here, i) is the image where the red fluorescence and bright field signals are merged, ii) red fluorescence alone, and iii) bright field alone. F: Confocal z-stack images are compiled to show the three-dimensional distribution of TSLs in the intracellular space.

liposomes also comprised of a lipid which was covalently attached to a red dye (DiI). The cells 13 ACS Paragon Plus Environment


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were incubated with liposomes for 4 hours before HIFU exposure. We have performed confocal studies that indicate that the liposomes are taken up by the cells within 2 hours of incubation (data not shown). To assess the liposome stability, we incubated the liposomes in 10% FBS (in PBS) and 50% FBS (in PBS) for 2.5 hours and imaged them by cryo-TEM in the supporting information section S1. The liposomes in 50% FBS remain stable for at least 2.5 hours.

HIFU reduces the therapeutic dose of Sorafenib in TSLs Figure 3 illustrates the effect of 8.7W HIFU on the viability of DU145 cells incubated with TSLs containing different concentration of Sorafenib. The cells treated with Sorafenibloaded TSLs but not exposed to HIFU showed a reduction in viability with time and Sorafenib dose (SFTSL bars in Figure 3). However, their viability at three days after treatment was still high (36 ± 5.5%) at the highest Sorafenib concentration used (50 µM). A decrease in the Sorafenib concentration to 20 µM and then to 10 µM increased the cell viability to 49 ± 1.9% and 63 ± 11%, respectively. The cells treated with both Sorafenib-loaded TSLs and HIFU (8.7W+SFTSL bars in Figure 3) showed a viability of 54 ± 1.3% at just 2 h post HIFU exposure and the lowest concentration of Sorafenib used (10 µM). The viability of the cells in this combined treatment group dropped to 15 ± 0.21% at 24 h and 9.6 ± 0.31% at 72 h. This change was statistically significant (p < 0.001). At three days post combined treatment, an increase in the Sorafenib concentration to 20 µM or 50 µM resulted in viabilities below measurable values, i.e., all the prostate cancer cells were essentially eradicated. When comparing 72 h data between the individual and combined treatment groups, HIFU led to a viability of 9.6 ± 0.31% at a low dose of Sorafenib (10 µM), which was significantly (p < 0.001) lower than the viability of cells


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treated with Sorafenib-loaded TSLs alone (36 ± 5.5%) at a high dose of Sorafenib (50 µM). Sorafenib at a concentration of 10 µM was used in remaining experiments to better understand the effect of HIFU power on prostate cancer cells.

*** **

100

Cell viability (%) normalized to control

2hr 24hr 72hr

*** ***

** p

Ablative Focused Ultrasound Synergistically Enhances Thermally

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