Accepted Manuscript

Title: Effect of HIFU Treatment on Tumor Targeting Efficacyof Docetaxel-loaded Pluronic Nanoparticles

Author: Keun Sang Oh Hyounkoo Han Byeong Deok YoonMinae Lee Hyuncheol Kim Dong Wan Seo Jae Hong SuhKwangmeyung Kim Ick Chan Kwon Soon Hong Yuk

PII: S0927-7765(14)00233-1DOI: http://dx.doi.org/doi:10.1016/j.colsurfb.2014.05.007Reference: COLSUB 6412

To appear in: Colloids and Surfaces B: Biointerfaces

Received date: 13-3-2014Revised date: 2-5-2014Accepted date: 3-5-2014

Please cite this article as: K.S. Oh, H. Han, B.D. Yoon, M. Lee, H. Kim, D.W.Seo, J.H. Suh, K. Kim, I.C. Kwon, S.H. Yuk, Effect of HIFU Treatment on TumorTargeting Efficacy of Docetaxel-loaded Pluronic Nanoparticles, Colloids and SurfacesB: Biointerfaces (2014), http://dx.doi.org/10.1016/j.colsurfb.2014.05.007

This is a PDF file of an unedited manuscript that has been accepted for publication.As a service to our customers we are providing this early version of the manuscript.The manuscript will undergo copyediting, typesetting, and review of the resulting proofbefore it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers thatapply to the journal pertain.

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Manuscript for Colloids and Surfaces B: Biointerfaces1

2

3

Effect of HIFU Treatment on Tumor Targeting Efficacy of Docetaxel-loaded Pluronic 4

Nanoparticles5

6

Keun Sang Oha, Hyounkoo Hanb,e, Byeong Deok Yoon a, Minae Lee a, Hyuncheol Kimb, Dong 7

Wan Seoc, Jae Hong Suhd, Kwangmeyung Kime, Ick Chan Kwone and Soon Hong Yuka,d*8

9

10aCollege of Pharmacy, Korea University, 2511 Sejongro, Sejong 339-700, Republic of Korea11

bChemical and Biomolecular Engineering, Sogang University, 35 Baekbeom Ro, Mapo-gu, 12

Seoul 121-742, Republic of Korea13

cDepartment of Internal Medicine, Asan Medical Center, University of Ulsan, College of 14

Medicine, 388-1 Pungnap-dong, Songpa-gu, Seoul 138-736, Republic of Korea15

dBiomedical Research Center, Korea University Guro Hospital, 148-1 Gurodong-ro, Guro-gu, 16

Seoul 152-703, Republic of Korea17

eCenter for Theragnosis, Korea Institute of Science and Technology, 39-1 Hawolgok-dong,18

Sungbuk-gu, Seoul 136-791, Republic of Korea19

20

21

* Corresponding authors: Soon Hong Yuk, Ph.D. (E-mail: shyuk@korea.ac.kr)22

23

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Abstract23

Numerous studies have been performed to identify the microenvironment of solid 24

tumors, which is responsible for the insufficient delivery of anticancer drugs to tumor cells 25

due to the poorly organized vasculature and the increased interstitial fluid pressure. As a 26

result, the extravasation of convection-dependent agents including NPs is severely limited. 27

Therefore, we have demonstrated the feasibility of targeting an enhancement of docetaxel-28

loaded Pluronic nanoparticles (NPs) using high-intensity focused ultrasound (HIFU) as an 29

external stimulus-induced clinical system in tumor tissue. The efficient extravasation of NPs 30

into the interior cells in tumor tissue was induced by relatively low HIFU exposure without 31

apparent acute tissue damage. The enhanced targeting of NPs with near-infrared fluorescence 32

dye was observed in tumor-bearing mice with various HIFU exposures. As a result, the 33

greatest accumulation of NPs at the tumor tissue was observed at an HIFU exposure of 20 34

W/cm2. However, the tumor tissue above at 20 W/cm2 appeared to be destroyed and the35

tumor targetability of NPs was significantly decreased owing to thermal ablation with 36

necrosis, resulting in the destruction of the tumor tissue and the blood vessels. In particular, a37

cross-sectional view of the tumor tissue verified that the NPs migrated into the middle of the 38

tumor tissue upon HIFU exposure. The preliminary results here demonstrate that HIFU 39

exposure through non-thermal mechanisms can aid with the extravasation of NPs into the40

interior cells of tumors and increase the therapeutic effect in enhanced and targeted cancer 41

therapy.42

43

44

Keywords: Targeting enhancement; ultrasound-induced; extravasation; pluronic45

nanoparticles; effective cancer therapy46

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1. Introduction47

Numerous studies have been conducted in an effort to identify the microenvironment of 48

solid tumors which is responsible for the insufficient delivery of anticancer drugs to tumor 49

cells [1,2]. Due to the poorly organized vasculature and a lack of functional lymphatics, 50

increased interstitial fluid pressure is generated in tumor tissue compared to normal tissue. As 51

a result, the extravasation of convection-dependent agents, including NPs, is severely limited 52

[3,4]. In particular, the extravasation of drug-loaded NPs is further exacerbated by the 53

presence of extracellular matrix (ECM), which is denser and more highly cross-linked than 54

healthy tissue [5,6].55

Recently, high-intensity focused ultrasound (HIFU) has been used effectively to ablate 56

solid tumors, where relatively long, continuous exposures are employed [7]. HIFU has also 57

been used noninvasively to enhance the local delivery of various macromolecules into 58

different tissue types by decreasing the temporal average intensities with shorter pulses and 59

relatively short duty cycles [8-10]. Many studies have reported that the acoustic energy of 60

focused ultrasound can induce reversible blood-brain barrier (BBB) disruption without 61

apparent acute tissue damage at the target site [11-15], and the ability of ultrasound to 62

permeabilize blood-tissue barriers makes it a potential method for the targeted drug delivery 63

of drugs, antibodies or genes. There are many different reports about changes in the degree of64

vessel permeability according to ultrasound-induced membrane porosity; moreover, the 65

duration of BBB disruption can transiently last up to 4 hours after sonication [16,17].66

Due to the presence of hydrophilic PEO chains on the surface, PEGylated NPs were 67

shown to have an increased circulation time in the body. In addition, they are believed to 68

accumulate in tumors due to the enhanced permeability and retention effect (EPR effect) [18-69

19]. However, it was also reported that the targeting of this type of NPs could be hindered by 70

the microenvironment of the solid tumor, as described above. This can be overcome with71

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HIFU, which may increase the permeability of the vascular endothelial cells, allowing the 72

NPs to escape the vascular space into the interstitial space of the tumor and aiding with the 73

distribution of the chemotherapeutic agent into the tumor, as schematically described in Fig. 74

1 [10,20].75

Pluronics are the tri-block copolymers of poly (ethylene oxide)-poly (propylene oxide)-76

poly (ethylene oxide) (PEO-PPO-PEO) of different molecular weights. Through the self-77

assembly of Pluronics in aqueous media, Pluronics form a micelle which is composed of an 78

inner hydrophobic core formed by PPO chain blocks and an outer hydrophilic shell formed 79

by nontoxic and non-immunogenic PEO blocks [21,22]. Using a combination of two 80

Pluronics, specifically Pluronic L61 and Pluronic F127, to form a Pluronic micelle, a 81

doxorubicin (DOX) formulation (SP1049C) was designed, demonstrating that SP1049C is 82

highly effective against multidrug-resistant cells [23,24]. A new type of Pluronic-based 83

nanoparticles (NPs) was formed in the melt state of a Pluronic/polyethylene glycol (PEG) 84

mixture through a temperature-induced phase transition. The PEG core containing paclitaxel 85

(PTX) was stabilized by Pluronic F-68 to form core/shell NPs, which showed enhanced 86

antitumor efficacy compared with a Cremophor EL-based PTX formulation [25,26]. Through 87

conjugation with rabies virus glycoprotein, the brain-targeted delivery of a protein was also 88

successfully demonstrated with Pluronic-based NPs, showing that this method can be utilized 89

for the treatment of brain diseases such as tumors, Alzheimer’s disease and Parkinson’s 90

disease [27]. 91

In this study, a targeting enhancement through the increased extravasation of docetaxel 92

(DTX)-loaded Pluronic NPs was demonstrated with HIFU exposure. To verify the feasibility 93

of the DTX-loaded Pluronic NPs as a delivery system for chemotherapy using drug-loaded 94

NPs with HIFU exposure, we evaluated the drug release patterns and in vivo bio-distribution95

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and conducted a histological analysis (TUNEL assay) of the Pluronic NPs with and without 96

HIFU exposure.97

98

2. Materials and methods99

2.1. Materials100

Pluronic F-68 (Pluronics, poly (ethylene oxide)-poly (propylene oxide)-poly 101

(ethylene oxide) triblock copolymer (Mw = 8,350; (EO)79(PO)28(EO)79) was obtained from 102

BASF Corp., Korea, and was used as received. Docetaxel (DTX, anhydrous form) was 103

purchased from Parling Pharma Tech Co., Ltd. (Shanghai, China). Tween 80 and soybean oil 104

were purchased from Sigma (St. Louis, MO, USA). The monoreactive hydroxysuccinimide 105

ester of Cy5.5 (Cy5.5-NHS) was obtained from Amersham Bioscience (Piscataway, USA).106

107

2.2. Preparation of the Pluonic NPs108

The DTX-loaded Pluronic NPs were prepared by a temperature-induced phase 109

transition. 300 mg of soybean oil/Tween 80 mixtures (weight ratio: 0.1) and 20 mg of DTX 110

were mixed to form a homogeneous drug-loaded core phase, which was subsequently mixed 111

with 200 mg of Pluronic F-68. The loading amount of DTX in the NPs was 3.84 wt. %. As 112

the temperature was increased to 60 °C, the mixture melted into a liquid phase. Equilibrium 113

was maintained at 60 °C for 10 minutes with stirring, and the liquid mixture was then cooled114

to 0 °C for about 10 minutes until it turn into a paraffin-like solid phase to induce a phase 115

transition. To evaluate the physicochemical characteristics of the DTX-loaded Pluronic NPs, 116

we added a pre-determined amount of distilled-deionized water or phosphate buffered 117

solution (PBS, pH 7.4) to the dried sample.118

To visualize in vivo tumor targetability using a NIR fluorescence imaging system, the 119

DTX-loaded Pluronic NPs were labeled with NIR dye cyanine 5.5 (Cy5.5). For this purpose, 120

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1 mg of Cy5.5 was mixed with a drug-loaded phase and subsequently mixed with Pluronic F-121

68 before the temperature induced phase transition.122

123

2.3. Particle size and morphology of the NPs124

The average diameter and size distribution of NPs (1 mg/mL of the NPs dispersed in125

phosphate buffered saline (PBS, pH 7.4)) were measured via dynamic light scattering (Zeta 126

Sizer Nano Series) at 632.8 nm and 25 °C. Transmittance electron microscopy (TEM) 127

measurement images were also taken to observe the morphology of Pluronic NPs. For the 128

TEM measurement, the freeze-dried NPs were dispersed in distilled-deionized water to obtain 129

a solution of 0.1 wt. %. To prepare a given sample, each solution was dropped on a carbon-130

coated grid and then dried at 25 °C in a vacuum oven for 24 hours. Samples were examined 131

with a Hitachi 7600 microscope operated at 100k V.132

133

2.4. In vitro drug release characteristics of the DTX-loaded Pluronic NPs134

To measure the release pattern of DTX from the Pluronic NPs, 10 mg of the NPs was 135

dispersed in 10 mL of PBS and put into a dialysis bag (MWCO: 500,000, Spectrum®, Rancho 136

Dominquez, CA), which was immersed in 15 mL of PBS, containing 0.1 % (w/v) Tween 80.137

The experimental setup was placed in a shaking water bath maintained at 37 °C and shaken 138

horizontally at 100 rpm. At predetermined time intervals, 2 mL aliquots of the release 139

medium (PBS) were withdrawn, and the total release medium was replaced with 15 ml of 140

fresh PBS to maintain the sink conditions. The quantification of released DTX was 141

determined by reverse-phase high performance liquid chromatography (RP-HPLC) using a 142

Capcell-pack C18 column and an acetonitrile/water (55/45, v/v) mobile phase over 15 minutes143

at a flow rate of 1.0 mL/min. The eluent was monitored by UV absorption at 227 nm.144

145

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2.5. Cytotoxicity and cellular uptake behavior of the Pluronic NPs146

Murine squamous cell carcinoma (SCC-7) cells were cultured in RPMI 1640 (Gibco, 147

Grand Island, NY) containing 10 % (v/v) FBS (Gibco) and 1 % (w/v) penicillin-streptomycin 148

at 37 °C in a humidified 5 % CO2-95 % air atmosphere. The cytotoxicity of free DTX149

(commercial DTX formulation (Taxotere®)), empty NPs, and the DTX-loaded Pluronic NPs 150

was evaluated using a MTT assay. The cells were seeded at a density of 5×103 cells/well in 151

96-well flat bottomed plates, and allowed to adhere overnight. The cells were washed twice 152

with PBS and incubated for 24 hours with various concentrations of free DTX, empty NPs, 153

and DTX-loaded Pluronic NPs. The cells were then washed twice with PBS to eliminate the 154

remaining drugs. 25 L of MTT solution (5 mg/mL in PBS) was added to each well and the 155

cells were incubated further for 2 hours at 37 °C. The cells were added and dissolved in 200 156

L of DMSO. Absorbance at 570 nm was measured with a microplate reader (VERSAmax™, 157

Molecular Devices Corp., Sunnyvale, CA). Cell viability was calculated as a percentage 158

compared to the culture medium only (negative control groups). Free DTX and the empty 159

NPs (NPs without DTX) were tested in parallel (equivalent DTX concentrations, and the160

empty NPs with the same NPs concentrations, respectively).161

To verify the cellular uptake of Cy5.5-/DTX-loaded Pluronic NPs, SCC-7 tumor162

cells (1×104 cells) were seeded onto a dish with cover slip and allowed to attach for 1 day. 163

After cell attachment, the medium was replaced with 2 mL of serum with a culture medium 164

containing the Cy5.5-/DTX-loaded Pluronic NPs (5 mg of DTX) and then incubated for 1 165

hour. The cells were then washed twice with PBS (pH 7.4) and fixed with a 4 % 166

paraformaldehyde solution. For nuclear staining, the cells were incubated with DAPI (3 167

mmol) for 5 minutes at 25 °C, following several washes in PBS. The intracellular localization 168

of Cy5.5-/DTX-loaded Pluronic NPs was observed using an IX81-ZDC focus drift 169

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compensating microscope (Olympus, Tokyo, Japan), where the excitation and emission 170

wavelengths were 673 nm and 692 nm. IC50 was calculated from the cell viability data as the 171

drug concentration in which cell growth was inhibited by 50 %.172

173

2.6. HIFU exposures for enhanced and targeted deliveryof the DTX-loaded Pluronic NPs174

The HIFU system used in the study and the method by which the tumors were treated 175

were previously described [28]. A commercially clinical available HIFU system (FEP-BY02,176

Yuande Bio-Medical Engineering, Beijing, China) was used to generate pulsed HIFU. Briefly, 177

the therapeutic procedure was guided by real-time US. A DU3 US imaging device (Esaote, 178

Genova, Italy) was used. This imaging probe is located in the center of the high intensity 179

focused ultrasound transducer. Ultrasound energy was produced by a transducer with a 5 x 5180

cm diameter and a focal length of 10 cm operated at a frequency of 0.8 MHz. Coupling of the 181

HIFU device to the skin was accomplished by warmed (37 °C) and degassed water. The 182

HIFU treatment was performed under intraperitoneal anesthesia with a solution containing 8 183

mg/mL ketamine (Ketalar®, Panpharma, Fougères, France) and 0.8 mg/mL xylazine 184

(Rompun®, Bayer Pharma, Puteaux, France) at 0.015 mL/g of body weight. After complete185

anesthesia was induced, the mice were carefully positioned on the left leg (tumor-bearing 186

site) so that the skin overlaying the lesion to be treated could be easily put in contact with the 187

degassed water. A vertical scanning mode was chosen and a line scan was used in the therapy. 188

The distance between slices was 5 mm and the treatment power was increased stepwise after 189

starting, and the ablation was terminated after the increased grey scale covered the tumor 190

margin. Exposure power of 10, 20, 30, and 80 W/cm2 was used. A typical exposure for an 191

entire tumor was within approximately 10 to 20 seconds. Prior work with these exposure 192

variables showed temperature elevations in SCC-7 tumor cells on the order of 4 °C to 5 °C.193

194

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2.7. In vivo biodistribution and tumor-targeting capability of the Pluronic NPs195

For in vivo experiments, SCC-7 (squamous cell carcinoma) cells were induced in 196

male C3H/HeN mice (5.5 weeks old, ORIENT BIO Inc. Korea) by means of a subcutaneous 197

injection of 1.0 x 106 cells suspended in a cell culture medium (RPMI 1640, 10 % fetal 198

bovine serum, 1 % antibiotic agent). When the tumor volume reached approximately 250 199

mm3 to 300 mm3, the mice received intravenous injection of the Cy5.5-/DTX-loaded Pluronic 200

NPs with 10 mg of DTX/kg with and without HIFU exposure. HIFU exposure was initiated 201

after intravenous injection of the NPs. To measure the tissue distribution, the time-dependent202

excretion profile, and the tumor-targeting capability, we positioned each animal on an 203

eXplore Optix system (Advanced Research Technologies Inc., Montreal, Canada). The mice 204

under an anesthetic state by inhalation of Gerolan sol. (enflurane as the active agent, Choong 205

Wae PHARMA Co., Korea) were automatically moved into the imaging chamber for 206

scanning. Laser power was optimized at 6 W, and the count time was set to 0.3 s per point. 207

Excitation and emission spots were raster-scanned in 1 mm steps over the selected region of 208

interest to generate emission wavelength scans. A 670 nm pulsed laser diode was used to 209

excite the Cy5.5 molecules. NIR fluorescence (NIRF) emission at 700 nm was collected and 210

detected with the aid of a fast photomultiplier tube (Hammamatsu, Japan) and a time-211

correlated single photon counting system (Becker and Hickl GmbH, Berlin, Germany). The 212

in vivo characteristics of Cy5.5-/DTX-loaded Pluronic NPs were confirmed by measuring the 213

NIRF intensity in the SCC-7 tumor-bearing mice (n=3 mice per group). Data were calculated 214

by using the region of interest (ROI) function of the Analysis Workstation software 215

(Advanced Research Technologies). To compare the tissue and tumor distributions of the 216

Cy5.5-/DTX-loaded Pluronic NPs, the mice were sacrificed 3 days post-injection. The major 217

organs including the liver, lung, spleen, kidney, and heart, as well as the tumor, were 218

dissected from the mice, and their fluorescence intensities were determined by using a 12-bit 219

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CCD camera (Image Station 4000 MM; Kodak, New Haven, CT) equipped with a special C-220

mount lens and a Cy5.5 bandpass emission filter (680-720 nm; Omega Optical). Identical 221

illumination settings (e.g., lamp voltage, filter, exposure time) were used in all animal 222

imaging studies. Quantification of in vivo tumor target specificity was verified as the total 223

photons per centimeter squared per steradian (p/s/cm2/sr) per each tumor (n=3 mice per224

group). All animal studies were carried out in accordance with the guidelines for animal 225

experiments at the Korea Institute of Science and Technology, Republic of Korea. To further 226

evaluate the distribution of the Cy5.5-/DTX-loaded Pluronic NPs in the tumor tissues, the 227

excised tumors were embedded in optimum cutting temperature tissue compound (OCT 228

compound, Sakura, Tokyo) for cryo-section. The 8 m thick frozen sections were not stained 229

with any dyes and were observed with fluorescence microscopy at 100 x magnification and 230

the number of apoptotic cells was counted in 4 random fields (100 x).231

232

2.8. Ex vivo histological analysis of the Pluronic NPs.233

After various HIFU exposures, tumors were removed at 24 hours after treatment and 234

fixed in 10 % buffered formalin (pH 7.4) and embedded in paraffin for hematoxylin-eosin 235

(H&E) and terminal deoxynucleotidyl transferased UTP nickend labeling (TUNEL). The 236

paraffin blocks were cut into 5 m tissue sections. Hematoxylin-eosin (H&E) staining was 237

used to observe the inflammatory reaction and morphology of tumor tissue. TUNEL staining 238

was performed using an ApopTag® Peroxidase In Situ Apoptosis Detection Kit 239

(Chemicon/Millipore, Billerica, MA) according to the manufacturer's protocol for observing 240

the apoptosis of tumor tissue by its effects despite short therapeutic time.241

242

2.9. Statistical analysis.243

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The data are expressed as the means ± SD of at least three experiments. The data for 244

in vivo characteristics were calculated using the region of interest (ROI) function of the 245

Analysis Workstation software (ART Advanced Research Technologies Inc., Montreal, 246

Canada). All data processing was performed using the ORIGIN® 8.0 statistical software 247

program (OriginLab Corp., Northampton, MA, USA). Statistical analysis was carried out 248

using one-way ANOVA and in all cases, a p value < 0.05 was considered significant, as249

noted in figures with asterisks.250

251

3. Results 252

3.1. Preparation and characterization of the Pluronic NPs253

The DTX-loaded Pluronic NPs were prepared in a molten mixture of DTX dissolved 254

in soybean oil/Tween 80 mixtures and Pluronic F-68 through a temperature-induced phase 255

transition at 60 °C for 20 minutes. Pluronic F-68 showed a melting transition at around 55 °C256

and the mixtures composed of soybean oil/Tween 80 and Pluronic F-68 showed a melting 257

transition at around 48 °C [29]. Therefore, we anticipated that the mixture composed of DTX258

dissolved in soybean oil/Tween 80 mixtures and Pluronic F-68 would melt into a 259

homogeneous solution at about 60 °C. The formation of DTX-loaded Plunonic NPs was 260

observed when the temperature was decreased to 0 °C. The morphology and size distribution 261

of Pluronic NPs were examined by TEM and a particle size analyzer, respectively (Fig. 2A).262

The Pluronic NPs had a spherical form with approximately 178 ± 24.67 nm diameters and 263

showed a typical sustained release pattern observed in drug release from a homogeneous 264

polymer matrix (Fig. 2B).265

266

3.2. Physicochemical properties of the Pluronic NPs267

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The cytotoxicity of Pluronic NPs was observed using a MTT assay. As a control, free 268

DTX was used under the same conditions. As expected, the empty Pluronic NPs exhibited 269

almost 100 % cell viability at a concentration of 500 g/mL. Even at a higher concentration 270

of 1,000 g/mL, the empty Pluronic NPs showed 90 % cell viability, indicating excellent 271

biocompatibility in the cell culture system. Also, free DTX and the DTX-loaded Pluronic272

NPs showed drug concentration-dependent cytotoxicity in the cell culture system. When the 273

DTX concentration was higher than 0.1 g/mL, the cytotoxicity of Pluronic NPs was lower 274

than that of free DTX. Less than 40 % of the SCC-7 tumor cells were viable after 2 days of 275

exposure to 1 g/mL of free DTX, whereas more than 75 % of the cells survived under the 276

same concentration of DTX in the Pluronic NPs (Fig. 3A). The low cytotoxicity of Pluronic277

NPs might be due to the sustained release of PTX from the NPs. As shown in Table 1, the 278

IC50 values of free DTX and the DTX-loaded Pluronic NPs (50.072 ± 2.167, 812.38 ± 4.579 279

g/mL, respectively) also verified that the DTX-loaded Pluronic NPs was less cytotoxic than 280

free DTX.281

The Pluronic NPs can penetrate and be taken up by SCC-7 tumor cells through an 282

endocytosis process [30]. Therefore, we monitored the cellular uptake characteristics of 283

Pluronic NPs in a cell culture system after 60 minutes post-incubation. The Cy5.5-/DTX-284

loaded Pluronic NPs were incubated in SCC-7 tumor cells (Fig. 3B). When their sub-cellular 285

localization was examined under a fluorescent microscope, fluorescence from the Pluronic286

NPs was clearly observed in the cytoplasm but not in the nuclear compartment of SCC-7 287

tumor cells.288

289

3.3. Tumor targeting enhancement of the Pluronic NPs by HIFU exposure290

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Enhanced tumor targeting of the Pluronic NPs was monitored with and without the 291

HIFU exposure after intravenous injection of the Cy5.5-/DTX-loaded Pluronic NPs into 292

SCC-7 tumor-bearing mice using an NIRF optical imaging system. (Fig. 4) As a control, the293

NIRF signal of Pluronic NPs was measured at the tumor site without HIFU exposure. We 294

were able to distinguish tumors from the surrounding background tissue and the NIRF signal295

reached its maximum level 6 hours after injection. 296

Enhanced tumor targeting of the Pluronic NPs was observed with the HIFU297

exposure. After showing a maximum value at 20 W/cm2 (The following variables were 298

employed for exposure: transducer with a 5 x 5 cm, a focal length = 10 cm; pulse repetition 299

frequency = 0. 8 MHz; duty cycle = 10 % (100 ms ON and 900 ms OFF); slice distance= 5 300

mm), tumor targetability of the Pluronic NPs decreased.301

The enhancement of tumor targetability was further confirmed by ex vivo NIRF302

images of dissected major organs (liver, lung, kidneys, spleen, heart, and tumor), as shown in 303

Fig. 5a. With and without HIFU exposure, higher accumulations of the Pluronic NPs were304

observed at tumors compared with all organs. More significant enhancement of NIRF305

intensity in tumors, however, was observed with HIFU exposure (Fig. 5A and Table 2).306

Cross-sectional views of tumor tissue were examined to verify the extravasation of the 307

Pluronic NPs into the tumor tissue (Fig. 5B and Table 2). Without HIFU exposure, 308

background noise of NIRF (appeared as red color) was observed throughout the tumor tissue 309

and yellow color from the Cy5.5-/DTX-loaded Pluronic NPs was observed on the surface 310

area of the tumor tissue. However, with HIFU exposure, yellow color from the Cy5.5-/DTX-311

loaded Pluronic NPs became dominant in the middle of the tumor tissue.312

313

3.4. Ex vivo histological analysis of the Pluronic NPs314

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H&E staining of the excised tumors was performed after 24 hours post-treatment as 315

shown in Fig. 6a. The Pluronic NPs-treated tumors without HIFU exposure were used as a 316

control. Significant morphological change was not found below HIFU exposure of 20 W/cm2317

compared with the control. However, with HIFU exposure of 30 W/cm2, severe tissue318

damage and hemolysis were observed with inflammatory cells.319

TUNEL staining was also performed to determine whether HIFU exposure induced320

apoptosis of the cancer cells. After showing a maximum value at 20 W/cm2, the number of 321

apoptotic cells decreased as shown in Fig. 6B. This pattern was already observed in the 322

enhancement change of tumor targeting as a function of HIFU intensity.323

324

4. Discussion325

The objective of this study is to demonstrate the synergistic effect of HIFU exposure326

on targeting efficacy of DTX-loaded Pluronic NPs based on the EPR effect. If the anti-cancer 327

drug-loaded NPs circulate for sufficient time, they should accumulate preferentially in tumors328

based on the EPR effect. This is thought to be due to the leaky and disorganized 329

characteristics of the blood vessels around the tumor tissue, which allow the NPs of 330

appropriate size to escape from the blood vessel. With the application of HIFU at 20 W/cm2, 331

the ability of ultrasound to permeabilize blood-tissue barriers is expected and makes HIFU a 332

potential method for the targeted drug delivery of DTX. Therefore, substantially enhanced 333

antitumor efficacy is expected with reduced side effect. As presented in Figs. 3 and 4, the 334

DTX-loaded Pluronic NPs accumulated at tumor tissue and this new DTX formulation of the 335

Pluronic NPs may minimize the adverse effects of the current free DTX formulation 336

containing Tween 80 and ethanol [31]. Note also that a previous study by the authors showed 337

enhanced antitumor efficacy of the DTX-loaded Pluronic NPs, compared with commercial 338

DTX formulation (Taxotere®) [29].339

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In normal tissues, the hydrostatic pressure from vessel to tissue and the osmotic 340

pressure from tissue to vessels are balanced for the transfer of oxygen, nutrients and waste 341

into and out of the tissue. Low interstitial pressure is usually maintained in normal tissue by 342

the lymphatic system to eliminate excess fluid from the tissue. However, the balance between343

these forces is severely disrupted with the formation of tumors [32]. A dense and highly 344

cross-linked extracellular matrix is formed in tumor tissue and this leads to an increase in the 345

interstitial pressure with a reduction of hydraulic flow to the tumor periphery [33,34]. For 346

efficient treatment of cancer, drug-loaded NPs exit the tumor blood vessel and enter the 347

tumor interstitial space to access the site of action. However, due to the microenvironment of 348

tumors described above, the driving forces that would normally assist the extravasation of 349

NPs may be reduced.350

HIFU exposure has been used to noninvasively and nondestructively enhance the 351

delivery of a variety of systemically administered agents [35,36]. Similar enhancement on the352

extravasation of NPs is expected with HIFU exposure. As shown in Fig. 4, the enhanced 353

accumulation of Pluronic NPs on tumor tissue was observed with HIFU exposure. After 354

showing a maximum value at 20 W/cm2, the accumulation of Pluronic NPs at tumor tissue 355

decreased. HIFU is presently being used for thermal ablation and direct tumor destruction. 356

During thermal ablation, tumor cells are destroyed through necrosis [7]. Above HIFU 357

intensity of 30 W/cm2, tumor cells appeared to be destroyed through thermal ablation (Fig. 6).358

Because thermal ablation with necrosis resulted in destruction of tumor tissue, a decrease in 359

the tumor targetability of Pluronic NPs was observed. 360

The in vivo biodistribution study, which was performed with 20 W/cm2 HIFU 361

intensity, revealed that the greatest accumulation of Pluronic NPs at the tumor was observed 362

(see Figs. 4 and 5A). In particular, as shown in Fig. 5B, the cross-sectional view of tumor 363

tissue verified that the Pluronic NPs migrated into the middle of tumor tissue with HIFU 364

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exposure. Regarding a possible explanation of the manner by which the HIFU exposure 365

enhanced the therapeutic effect of injected agents, it has been suggested that HIFU exposure 366

enlarges the effective pore size in the tumor, leading to the better penetration and distribution 367

of injected agents [37, 38]. This is supported by the enhancement of tumor targetability of the 368

Pluronic NPs with HIFU exposure observed in the present study.369

HIFU exposures are clinically used in continuous mode to maximize the heat 370

generation for tissue ablation. However, for more delicate and non-thermal mechanisms, the 371

rate of energy disposition should be reduced. In the combination with Pluronic NPs, HIFU 372

exposure with 20 W/cm2 showed the highest number of apoptotic cells (see Fig. 6) indicating 373

that effective cancer treatment could be accomplished through a noninvasive approach with374

HIFU exposure.375

376

5. Conclusions377

Because of prolonged circulation of the Pluronic NPs in the blood, the DTX-loaded 378

Pluronic NPs are believed to be effective in targeted cancer therapy based on the EPR effect.379

However, with a more detailed understanding of the tumor microenvironment, elevated 380

interstitial tumor pressure and the dense tumor extracellular matrix have been recognized as 381

formidable barriers to the extravasation of NPs. Enhanced targeting at tumor and effective 382

extravasation into interior cells in the tumor tissue were demonstrated using the Pluronic NPs 383

with HIFU exposure. This approach can transiently increase the effective pore size of tumor 384

tissue with the enhanced permeability of Pluronic NPs through non-thermal mechanisms. 385

This was confirmed by observing the in vivo biodistribution of Pluronic NPs with HIFU 386

exposure. The preliminary results in this study demonstrate that HIFU exposure through non-387

thermal mechanisms can aid the extravasation of NPs into interior cells in tumors and 388

increase the therapeutic effect in targeted cancer therapy.389

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Acknowledgments

This work was supported by the National Research Foundation (NRF) of Korea grants

funded by the Korean government (MEST) (20110027932, 2012028831 and 2013063969).

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Figure Captions

Fig. 1. (A) Schematic description of enhanced tumor targeting with HIFU exposure and (B)

expected behavior of the Pluronic NPs in the vessel.

Fig. 2. (A) The size distribution and morphology of DTX-loaded Pluronic NPs and (B) in

vitro release behavior of DTX in the Pluronic NPs. Data are presented as the mean ± SD

(n=4).

Fig. 3. (A) The cellular uptake of Cy5.5-/DTX-loaded Pluronic NPs with SCC-7 tumor cells

and (B) in vitro cytotoxicity at different concentrations of free DTX, the empty NPs and the

DTX-loaded Pluronic NPs. Data are presented as the means ± SD (n=6).

Fig. 4. (A) Damage assessment of tumor in mice treated with different HIFU exposure

intensities, (B) in vivo whole body non-invasive biodistribution and (C) fluorescence

quantification analysis of localized the Cy5.5-/DTX-loaded NPs at tumor using in vivo NIRF

imaging system.

Fig. 5. (A) Representative ex vivo fluorescence images of dissected major organs (liver, lung,

spleen, kidney, heart and tumor) and (B) cross-sectional images of excised tumor tissues of

sacrificed SCC-7 tumor-bearing mice after 24 hours with and wiout HIFU exposure.

Fig. 6. (A) Representative histological images and (B) quantification data of apoptotic cells

of sliced tumor tissues with different HIFU exposure intensity, stained using hematoxylin and

eosin (H&E) and TUNEL assay. Tumors were excised at 24 hours after HIFU exposure. Data

are presented as the means ± SD (n=4).

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Graphical abstracts

DTX-loaded Pluronic NPs Pluronic F-68

(PEO-PPO-PEO tri-block copolymer)

Docetaxel (anti-cancer drug)

Soybean oil/Tween 80 mixtures

Ultrasound-induced targeting enhancement by

HIFU (high-intensity focused ultrasound)

exposure

Local site

(Tumor)

TransducerHIFU beam

Tumor TissueNormal Tissue

Free Drug Passive Targeting HIFU-triggered Targeting

Angiogenic

vessel

Untreated

HIFU10 W/cm2 20 W/cm2 30 W/cm2

3hr

6hrs

24hrs

80 W/cm2

Graphical Abstract (for review)

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Figure 1.

A

B

Figure(s)

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Figure. 2

A

0 100 200 300 4000

20

40

60

80

100

Inte

nsi

ty

Diameter (nm)

B

0 12 24 36 48 60 72

0

20

40

60

80

100

Rele

ase

d A

mo

un

t o

f D

TX

(%

)

Time (hrs)

Free DTX

DTX-loaded Pluronic NPs

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Figure. 3

A

B

0.1 1 10 100 500 10000

25

50

75

100

125

Ce

ll v

iab

ilit

y (

%)

Concentration (g/mL)

Empty NPs

0.1 1 10 100 500 10000

25

50

75

100

125

Ce

ll v

iab

ilit

y (

%)

Concentration (g/ml)

Free DTX

DTX-loaded Pluronic NPs

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Figure. 4

A

B C

0 3 6 9 12 15 18 21 240.0

5.0x103

1.0x104

1.5x104

2.0x104

2.5x104

Untreated

10 W/cm2

20 W/cm2

30 W/cm2

80 W/cm2

Av

era

ge

no

ma

lize

d c

ou

nte

r (N

C)

Time (hours)

Untreated

HIFU10 W/cm2 20 W/cm2 30 W/cm2

3hr

6hrs

24hrs

80 W/cm2

Tumor

Untreated 10 W/cm2 20 W/cm2 30 W/cm2 80 W/cm2

Tumor

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B Control Untreated 20 W/cm2

Untreated

20 W/cm2

A

Figure. 5

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B

Figure. 6

A

0 (Untreated) 10 20 300

20

40

60

80

100

Nu

mb

er

of

TU

NE

L-p

os

itiv

e

ap

op

toti

c c

ell

s/f

ield

(0

.5 m

m2)

HIFU power (W/cm2) * : p value < 0.05

** : p value > 0.05

*** *

Untreated 10 W/cm2

x 400

20 W/cm2 30 W/cm2

H&E staining

TUNEL assay

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Table 1

Table 1. The IC50 value of free DTX, empty NPs, and the DTX-loaded Pluronic NPs

after 24 hours incubation with SCC-7 cancer cells.

Samples IC50 (µg/mL)

Free DTX 50.072 ± 2.167

Empty NPs -

DTX-loaded Pluronic NPs 812.38 ± 4.579

Data are presented as the means ± S.D. (n=6).

Table(s)

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Table 2

Table 2. Fluorescence quantification data of in vivo biodistribution of the DTX-loaded

Pluronic NPs with and without HIFU exposure.

Organ Signal Intensity (Fig. 5A)

Untreated 20 W/cm2

Liver 1028 ± 78.89 1065 ± 64.08

Lung 689 ± 43.52 647 ± 57.46

Spleen 611 ± 38.43 672 ± 48.51

Kidney 679 ± 39.47 738 ± 51.07

Heart 492 ± 31.08 551 ± 48.24

Tumor 3046 ± 145.61 4756 ± 122.43

Tumor/Liver

Tumor/Kidney

2.963

4.486

4.504

6.444

Samples Signal Intensity (Fig. 5B)

Control

(Normal tumor tissue) N.D.

Untreated 568.2505 ± 12.457

20 W/Cm2

865.0514 ± 14.534

Cy5.5-/DTX-loaded Pluronic NPs were recorded as total photon counts per centimeter

squared per steradian (p/s/cm2/sr) per each excised organ at 24 hours post-injection in SCC-7

tumor-bearing mice (n = 3 mice per group). All data presented as means ± S.D. (n = 3).

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Research Highlights

1. Targeting enhancement of docetaxel (DTX)-loaded Pluronic nanoparticles (NPs) was

demonstrated using high intensity focused ultrasound (HIFU) as an external stimuli-

responsive clinical system.

2. Efficient extravasation of the Pluronic NPs into the interior cells in the tumor tissue was

induced by the relatively low HIFU exposure where this can transiently increase the effective

pore size (endothelial cell tight junctions) of a tumor tissue with enhanced permeability of the

Pluronic NPs through non-thermal mechanisms.

3. To verify the targeting enhancement through the efficient extravasation, the biodistribution

of the Pluronic NPs was observed in in vivo animal model with various HIFU exposures.

*Highlights (for review)