Concurrent blood–brain barrier opening and local drug delivery using drug-carrying microbubbles and focused ultrasound for brain glioma treatment

Concurrent blood–brain barrier opening and local drug delivery using drug-carrying microbubbles and focused ultrasound for brain glioma treatment

Biomaterials 33 (2012) 704e712 Contents lists available at SciVerse ScienceDirect Biomaterials journal homepage: www.elsevier.com/locate/biomaterial...

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Biomaterials 33 (2012) 704e712

Contents lists available at SciVerse ScienceDirect

Biomaterials journal homepage: www.elsevier.com/locate/biomaterials

Concurrent bloodebrain barrier opening and local drug delivery using drug-carrying microbubbles and focused ultrasound for brain glioma treatment Chien-Yu Ting a,1, Ching-Hsiang Fan a,1, Hao-Li Liu b, **, Chiung-Yin Huang c, Han-Yi Hsieh b, Tzu-Chen Yen d, Kuo-Chen Wei c, Chih-Kuang Yeh a, * a

Biomedical Engineering and Environmental Science, National Tsing Hua University, No. 101, Section 2, Kuang-Fu Road, Hsinchu 30013, Taiwan, ROC Department of Electrical Engineering, Chang-Gung University, 259 Wen-Hwa 1st Road, Kuei-Shan, Tao-Yuan 33302, Taiwan, ROC Department of Neurosurgery, Chang Gung University College of Medicine and Memorial Hospital, 5 Fu-shing Road, Kuei-Shan, Tao-Yuan 33305, Taiwan, ROC d Department of Nuclear Medicine, Chang Gung University College of Medicine and Memorial Hospital, 5 Fu-shing Road, Kuei-Shan, Tao-Yuan 33305, Taiwan, ROC b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 16 September 2011 Accepted 29 September 2011 Available online 22 October 2011

Glioblastoma multiforme (GBM) is a highly malignant brain tumor. The bloodebrain barrier (BBB) provides a major obstacle to chemotherapy since therapeutic doses cannot be achieved by traditional drug delivery without severe systemic cytotoxic effects. Recently, microbubble (MB)-enhanced focused ultrasound (FUS) was shown to temporally and locally disrupt the BBB thereby enhancing drug delivery into brain tumors. Here we propose the concept of smart, multifunctional MBs capable of facilitating FUS-induced BBB disruption while serving as drug-carrying vehicles and protecting drugs from rapid degradation. The designed MBs had a high loading capacity (efficiency of 68.01  4.35%) for 1,3-bis(2chloroethyl)-1- nitrosourea (BCNU). When combined with FUS (1-MHz), these BCNU-MBs facilitated local BBB disruption and simultaneously released BCNU at the target site, thus increasing local BCNU deposition. Encapsulation of BCNU in MBs prolonged its circulatory half-life by 5-fold, and accumulation of BCNU in the liver was reduced 5-fold due to the slow reticuloendothelial system uptake of BCNU-MBs. In tumor-bearing animals, BCNU-MBs with FUS controlled tumor progression (915.3%e39.6%) and improved median survival (29 dayse32.5 days). This study provides a new approach for designing multifunctional MBs to facilitate FUS-mediated chemotherapy for brain tumor treatment. Ó 2011 Elsevier Ltd. All rights reserved.

Keywords: Glioblastoma multiforme (GBM) Focused ultrasound (FUS) Microbubble (MB) Chemotherapy Bloodebrain barrier (BBB)

1. Introduction Brain tumors have a low incidence but high lethality compared to other cancers. Glioblastoma multiforme (GBM) is the most common primary brain tumor and is highly malignant [1e4]. The current standard clinical treatment for GBM includes surgical resection, which is usually the first choice and is often combined with irradiation and chemotherapy [5,6]. However, the prognosis of patients with GBM remains poor with a medium survival of 1e2 years [5,7,8]. Moreover, most tumors recur locally within 2 cm of the original lesion [7,9,10]. Chemotherapeutic agents such as 1,3-bis(2-chloroethyl)-1nitrosourea (BCNU, Carmustine) are widely used in brain tumor

* Corresponding author. Tel.: þ886 3 571 5131x34234; fax: þ886 3 571 8649. ** Corresponding author. Tel.: þ886 3 211 8800x5677; fax: þ886 3 211 8026. E-mail addresses: [email protected] (H.-L. Liu), [email protected] (C.-K. Yeh). 1 C.-Y. Ting and C.-H. Fan contributed equally to this work. 0142-9612/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2011.09.096

treatment [7,11]. However, traditional intravenous (IV) administration of chemotherapeutic agents has several limitations such as systemic cytotoxic effects and limited drug penetration due to the bloodebrain barrier (BBB) [5,12] and bloodebrain tumor barrier (BTB) [13,14]. The high doses of toxic antineoplastic agents that are necessary to overcome the BBB for complete tumor control are usually accompanied by serious side effects such as pulmonary fibrosis, myelosuppression, and damage of hepatic and renal function [9,11,15]. Current strategies to overcome the BBB include osmotic or radiation-induced BBB disruption (BBBD) [12,16,17]. However, osmotic BBBD affects all brain tissue that is supplied by the artery used for infusion, resulting in penetration of the cytotoxic drug into non-targeted regions [18e20], whereas the required dose of radiation to induce BBBD can lead to severe damage [21]. Microbubble (MB)-enhanced focused ultrasound (FUS) is a proven strategy for non-invasive, transient, reversible and local BBBD [22] resulting in more precisely targeted drug delivery. In addition, numerous drug carriers such as polymeric assemblies, liposomes, and micelles with various controlled release methods have been developed as promising strategies to increase local drug

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concentration while reducing systemic side effects [5,9,11,15,22e24]. A major drawback of these combined strategies is the requirement for an IV injection of MBs to facilitate the BBBD, followed by a second IV administration of chemotherapeutic agent. Moreover, both the MBs and chemotherapeutics currently have short half-lives due to their uptake by the reticuloendothelial system (RES) when circulating through liver. Besides, BCNU has a half-life of about 20e50 min in vitro and less than 15 min in vivo, which further limits its efficacy after systemic application [15,25]. The purpose of this study was to develop smart, multifunction MBs capable of: (1) serving as high-payload drug (BCNU)-carrying vehicles thus reducing the requirement for IV injection from two to one; (2) facilitating BBBD during FUS exposure; and (3) extending drug half-life in circulation to reduce both the total injected chemotherapeutic dose and systemic toxicity [26] (Fig. 1). Here we describe the preparation and properties of BCNU-MBs, and their ability to disrupt the BBB and release the drug when combined with FUS, in vivo. We then evaluate the feasibility and treatment efficacy of this drug delivery system in an animal glioma model.

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FL, USA) with a 0.7e20 mm range. Smaller particles (<1 mm) were measured by dynamic light scattering (Nanosizer-S, Malvern, London, UK). BCNU-MBs and pure lipid MBs were visualized with an inverted optical microscope (IX-71, Olympus, NY, USA). Drug loading and encapsulation efficiency were analyzed using a reverse method by high performance liquid chromatography (HPLC) with a UV detector (L-2400, Hitachi, Tokyo, Japan). BCNU-MB formulations were centrifuged for 3 min at 6000 rpm (mini-micro centrifuge, BERTEC ENTERPRISE CO., LTD., Taiwan). The residual BCNU after BCNU-MB production (Wres) was determined after carefully separating the underlying liquid phase from the top foam cake with a syringe needle. Drug payload (WBCNU-MB) was calculated as the difference between the total amount of BCNU (Wtot) and Wres. Drug encapsulation efficiency was then calculated by as: Encapsulation efficiency (%) ¼ WBCNU-MB/Wtot  100%. The mobile phase solution consisted of 60% (v/v) HPLC-grade methanol in deionized water. A mightysil RP-18 GP 250e4.6 column (Kanto Chemicals, Tokyo, Japan) was used with a flow rate of 2.0 mL/min and detection wavelength of 237 nm [26]. The amount of BCNU was quantified by the area under its characteristic peak at 3.2 min retention time. 2.2.2. In vitro drug leakage Electrolytes can destroy electrostatic interactions, leading to dissociation and drug loss from MBs [28]. We determined leakage of BCNU from MBs, under various conditions. BCNU-MBs were incubated in PBS at 4  C or 37  C and periodically centrifuged. The residual clear liquid was collected to quantify the amount of BCNU leakage (Wleakage) by HPLC, as: BCNU leakage percentage (%) ¼ Wleakage/WBCNU-MB  100%.

2. Materials and methods 2.1. Preparation of BCNU-loaded MBs

2.3. In vitro FUS-induced BCNU release

BCNU-loaded MBs (BCNU-MBs) were prepared by the thin-film hydration method. Briefly, 1 mL bubble formulation contained DPPC (DipalmitoylPhosphatidyl-Choline) (Avanti Polar Lipids, AL, USA) and DSPE-PEG-2000 (1,2Distearoyl-sn-Glycero-3-Phosphoethanolamine-N-[Methoxy(polyethyleneglycol)2000]) (Avanti Polar Lipids) at a molar ratio of 19:1, with 37.6 mL BCNU solution (100 mg BCNU dissolved in 3 mL purified ethanol) (Bristol-Myers Squibb, NY, USA), blended into chloroform in a vial. The organic solvent was eliminated at 10  C under reduced pressure over 24 h with a rotary evaporator (Rotavapor R-210, BüchiLabortechnik AG, Flawil, Switzerland). The dried lipid-film was then hydrated with 5 mL/mL glycerol phosphate-buffered saline (PBS), and the gas in the vial was replaced with perfluoropropane (C3F8). Finally, the vial was mechanically shaken for 45 s using a homemade agitator. The hydrophobicity of the BCNU drug molecules allowed them to be embedded in the MBs where they attached to the phospholipid shell by both electrostatic and hydrophobic interactions (Fig. 2A). These BCNU-MBs retained all the important physical characteristics of pure lipid MBs for ultrasoundinduced destruction. Pure lipid MBs were prepared by the same method as BCNUMBs, but without adding BCNU.

A single-element 1-MHz FUS transducer (V302, Panametrics, Waltham, MA, USA; diameter ¼ 38 mm, focus length ¼ 60 mm) was used to trigger the release of BCNU from BCNU-MBs. A function generator (WW2571, Tabor electronics, Haifa, Israel) created sonication pulses which were amplified with a radio-frequency power amplifier (150A100B, AR, PA, USA) to drive the 1-MHz FUS transducer. A self-assembled external impedance matching circuit was used to match the electric impedance of the transducer with the output impedance of the amplifier. A polyvinylidene-difluoride type hydrophone (Onda, Sunnyvale, CA, USA) was used to measure and calibrate the axial and lateral acoustic fields generated by the 1MHz FUS transducer in a tank filled with degassed, distilled water. The halfmaximum pressure amplitude at the focal zone had a diameter of 3 mm and length of 26 mm. A flow phantom containing BCNU-MBs was constructed to estimate the destruction efficiency of BCNU-MB by 1-MHz FUS sonication. Two grams of agarose powder (UltraPureÔ Agarose, Invitrogen, CA, USA) were dissolved in 100 mL water. A tube with a 5 mm outer diameter was embedded inside the agar and withdrawn after congelation to form a flow chamber. BCNU-MBs were diluted 1000 times with 0.9% normal saline and injected into the flow chamber by syringe pump with a flow rate of 1 mL/h. The diluted BCNU-MBs were destructed by FUS with various sonication parameters (acoustic pressure: 0.3, 0.4, 0.5, 0.6, and 1.1 MPa; pulse repetition frequency (PRF): 1, 2, 5, and 10 Hz; number of cycles: 1000, 5000, and 10000), followed by collection of the sonicated samples from the other end of the chamber and quantification of the released BCNU by HPLC.

2.2. Characterization of BCNU-MBs 2.2.1. Size distribution, MB concentration, and drug payload The size distribution and concentration of MBs were determined with a Coulter counter equipped with a 30 mm sensor orifice (Multisizer 3, Beckman Coulter, Miami

Fig. 1. Summary of our hypotheses for drug-loaded MBs delivery into brain tissue, and controlled release triggered by FUS sonication. The blue circles represent drug-loaded MBs, and the red/yellow octagons represent the drug. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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2.4. In vivo studies 2.4.1. Animal preparation All animal procedures were performed according to the guidelines of the National Tsing Hua University animal committee. A total of 62 healthy male SpragueeDawley (SD) rats (250e300 g) (w/o tumor implantation) were used. Before the experiment, animals were anesthetized intraperitoneally with chloralhydrate (400 mg/kg). A craniotomy (approximately 1  1 cm2) was performed on each animal to allow the transmission of the ultrasound beam into the brain. A catheter (PE50, IntramedicÔ, Clay Adams Inc., NJ, USA) was inserted into the right jugular vein for injection of MBs, drugs, and dyes. Animal body temperature was maintained at 36  1  C by a heating pad (THM100, Indus Instruments, TX, USA) during the experiment. 2.4.2. Experimental setup The experimental apparatus consisted a 1-MHz FUS transducer used for transmitting sonication pulses and another single-element 25-MHz transducer (V324, Panametrics; diameter ¼ 6.1 mm, focus length ¼ 12.3 mm) used for imaging to guide therapy (Fig. 3A). The two transducers were arranged parallel to each other, approximately 34 mm apart, using an in-house manufactured holder. The focal points of the 1-MHz FUS and 25-MHz imaging beams were thus fixed at the same depth. The holder was mounted on a stage attached to a computer-controlled 2-D positioning system (HR8, Nanomotion, Yokneam, Israel). A motion controller (DMC-2140, Galil Motion Control, Rocklin, CA, USA) generated triggers to synchronize ultrasound pulsing and data acquisition based on optical encoder counts. Ultrasound images were acquired using a homemade ultrasound scanning system [29]. Briefly, the pulser/receiver (5900PR, Panametrics) provided impulse excitation to the 25-MHz transducer and received the radio frequency (RF) signals. The RF signal was amplified by a preamplifier (AU-1114-BNC, Miteq, Hauppauge, NY, USA) and then further amplified within the pulser/receiver. RF signals were digitized with a PC-based analog-to-digital board (PCI-9820, AdLink, Taiwan) and processed offline with MATLAB (The MathWorks, Natick, MA, USA). The 1-MHz FUS was moved to the sonication site by the 2-D motorized translation stage immediately after imageguidance by 25-MHz ultrasound in the B-mode. The animal was placed prone and directly under a water reservoir (4  4 cm2 window on the bottom, sealed with a polyurethane membrane to allow entry of

ultrasound) with its head attached tightly to the membrane window. A removable cone was used to direct the 1-MHz FUS beam into the brain. Ultrasound coupling gel (aquasonic 100, Parker laboratories, Fairfield, NJ, USA) was applied between the cranial window and the membrane to maximize the transmission of ultrasound between the transducer and the brain. The animal was secured on the stereotaxic apparatus with ear bars and a bite bar during the experiment. 2.4.3. BCNU-MB in vivo stability The in vivo life-time of BCNU-MBs was determined by evaluation of their echo intensity within brain tissues, using serial ultrasound B-mode images. BCNU-MBs and pure lipid MBs (control) were adjusted to the same concentration (1  109 MBs/mL) with 0.9% normal saline, and were administered separately by a bolus injection to construct a time-intensity curve (TIC). The life-time of MBs was then predicted from their clearance time. All data analyses were performed with MATLAB. 2.4.4. In vivo quantification of drug delivery Three animal-experiment groups were designed, all with a BCNU dose of 1.25 mg per animal. In the first group, the aim was to determine how the delivery of ultrasound affected the efficiency of drug release. Different levels of acoustic pressure (0.3, 0.4, 0.5, and 0.6 MPa), pulse repetition frequency (1, 2, 5, and 10 Hz), and number of FUS sonication locations (1, 2, and 4 locations) were attempted. Rats received 0.5 mL of BCNU-MBs diluted to 1 mL with 0.9% normal saline. After a 20-s long injection of BCNU-MBs, FUS was delivered into the left hemisphere of the brain. Animals were sacrificed 10 min after the experiment to minimize the loss of BCNU caused by hydrolysis. The brain tissue was harvested and divided into sonicated and non-sonicated hemispheres. These brain samples were homogenized and BCNU was extracted with 2 mL methanol by ultrasonic cleaner (2510, Branson Ultrasonics, Danbury, CT, USA) for 30 min. After drug extraction, the samples were centrifuged at 13,500 rpm for 10 min using a freezing centrifuge (SIGMA 3-30K, Heraeus Co., Germany). The clear supernatant was collected and the precipitate was re-extracted twice [27]. All of the supernatants were filtered with a 0.20 mm filter and finally analyzed by HPLC. Procedures were performed at 0e4  C to reduce BCNU degradation. In the second animal-experiment group, we aimed to compare and evaluate the therapeutic efficacies of BCNU, BCNU-MBs, and their combination with FUS therapy.

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Animals were divided into four subgroups: (1) Injection of BCNU-MBs combined with FUS in the left brain hemisphere (BCNU-MB þ FUS subgroup); (2) Injection of BCNU-MBs alone without FUS (BCNU-MB w/o FUS subgroup); (3) Injection of a single dose of BCNU alone (BCNU subgroup); and (4) BCNU injection following by pure lipid MBs combined with FUS ((MB þ FUS) þ BCNU subgroup). In subgroup (4), FUS was applied right after injection of 0.05 mL of pure lipid MBs diluted to 0.2 mL with normal saline to open the BBB, followed by injection of a single dose of BCNU. BCNU was extracted as detailed above. In the third experimental group, our aim was to evaluate whether the MB carrier could protect BCNU from degradation, and then release it by FUS after a prolonged circulation time. Animals were divided into the same four subgroups that were used in the second experiment. The drug extraction procedures were the same, with the following circulation and extraction times: In subgroup (1) BCNU-MBs were allowed to circulate for 30 min before FUS exposure to the left hemisphere of the brain. In subgroups (2e4), drugs were also allowed to circulate for 30 min. Pure lipid MBs diluted to 0.2 mL with 0.9% normal saline were then injected to open the BBB with FUS, only in subgroup (4). After an additional 10 min, animal brains from each subgroup were then removed to quantify the residual BCNU concentration.

a measure of vascular permeability. After finishing the experiments, animals were sacrificed and perfused with 0.9% normal saline via the left ventricle until colorless perfusion fluid appeared from the right atrium. The brain tissues were removed, rapidly frozen, embedded in optimal-cutting-temperature compound (Tissue-Tek, Sakura, CA, USA) and stored at 50  C. Tissues were sliced into coronal sections, and EB extravasation was visible by eye as blue stained brain tissue. Histological evaluations were performed by a person who was blinded to the ultrasound parameters but was informed of the specific sonicated brain side. 2.4.6. In vivo evaluation of cytotoxicity To evaluate temporal cytotoxic effects induced by different drug delivery, all of the liver tissues from the animals of the second experimental group were harvested (animals were sacrificed 10 min after the experiment) for biochemical analysis of liver function. Drug deposition in liver tissues was quantified like in the brain. The serum of animals under the same treatment procedures was also isolated 24 h after each treatment, and levels of AST (Aspartate aminotransferase) and ALT (Alanine aminotransferase) were determined to assess liver function. 2.5. Rat glioma model and treatment evaluation

2.4.5. Confirmation of (BCNU-MB)-enhanced FUS-BBBD Evans blue (EB) dye (100 mg/kg) (SigmaeAldrich, MO, USA) was administered 5 min before sonication to verify successful BBBD. The dye binds to albumin as

A total of 5  105 cells/mL C-6 glioma cells were implanted in male SD rats (200e250 g). The implant location was 0.5 mm anterior and 3 mm lateral to the

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bregma on the left side of the skull and at a depth of 4.5 mm from the brain surface (at the striatum) [27]. These tumor-embedded animals were randomly divided into three groups: control (N ¼ 6), BCNU alone (BCNU, N ¼ 7), and BCNUMBs combined with FUS (BCNU-MB þ FUS, N ¼ 6). It has recently been suggested that a more fractionated schedule of drug administration using smaller doses than the maximum tolerated dose may be equally or more effective than one dose [30]. Low-dose administration is also less toxic. Therefore, treatments were performed consecutively on day 4 and 5 after tumor implantation. FUS irradiation was applied transcranially with 0.7 MPa pressure, 10 ms burst length, a 5% duty cycle, a 5 Hz PRF, and two sites of sonication. Targeted sonication was delivered with 2-mm gaps between sonication sites and a duration of 1 min of sonication per site.

2.6. Magnetic resonance imaging (MRI) Tumor progression was assessed by MRI with a 3T scanner (Trio with Tim, Siemens, Erlangen, Germany) and a standard wrist coil with an inner diameter of 13 cm at one-week intervals, 10 days after tumor implantation (Fig. 3B). Animals were anesthetized with a mixture of oxygen (0.8 L/min at 1.0 Bar, 21  C) and 2% vaporized isoflurane using an anesthesia vaporizer throughout the MRI imaging process. Tumor volume was quantified by analyzing T2-weighted images with the following parameters: TR ¼ 3490 ms, TE ¼ 98 ms, matrix size ¼ 144  256, FOV ¼ 47  83 mm (resolution ¼ 0.3  0.3 mm), slice thickness ¼ 1 mm. Tumor volume data acquired at each time point were normalized to the volume obtained on day 10. Three-dimensional (3D) tumor construction was post-processed by AVIZO (VSG, MA, USA).

2.7. Statistics Results are presented as the mean and standard error of the mean of at least three independent measurements. All statistical evaluations including tumor volume, and BCNU uptake between the unsonicated side of the brain and the contralateral side were carried out with unpaired two-tailed Student’s t-tests. A pvalue of less than 0.05 (p < 0.05) was considered significant. Statistical analysis was performed by Microsoft Excel 2010 (Microsoft, NY, USA).

3. Results 3.1. Characterization of BCNU-MB MBs alone and BCNU-encapsulating MBs both showed uniform size distribution with a single peak and a range of 500 nm to 3 mm (Fig. 2B). The mean size of pure lipid MBs was 1.11  0.05 mm. After loading the drug into the MBs, the mean size of BCNU-MBs increased to 1.32  0.18 mm. The mean concentration of MBs was 27.67  2.42  109 MB/mL and 19.78  4.9  109 MB/mL for BCNUMBs. The amount of BCNU encapsulated in MBs increased linearly with Wtot from 0 to 2 mg, after which it plateaued (Fig. 4A). The average drug load was 1.67  0.34 mg/mL, and the drug encapsulation efficiency was 68.01  4.35%. BCNU retention was stable at 4  C (only 4.12  0.64% leakage after 5 h), but leakage dramatically increased at 37  C from 2.50  0.58% at 1 h to 15.12  0.33% at 3 h and nearly 100% after 24 h (Fig. 4B). The TICs from ultrasound images demonstrated that BCNU-MBs and pure lipid MBs had similar wash-in phases that reached their peaks almost at the same time and then decayed over time at rates that resembled each other (Fig. 4C). Thus, after drug loading, BCNUMBs appeared to retain good acoustic and physical properties similar to pure lipid MBs. Different sonication parameters including acoustic pressure, PRF and number of cycles were tested to optimize release of BCNU in the targeted FUS-exposure region (Fig. 4D; standard settings were cycle number ¼ 10000, PRF ¼ 5 Hz, and acoustic pressure ¼ 0.5 MPa). The drug releasing efficiency reached 64.66  0.56% at 0.5 MPa, and did not increase significantly at higher pressures. On the other hand, efficiency of drug release increased monotonically with PRF, reaching 68.91  3.68% at 10 Hz. Increasing

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the cycle number did not affect BCNU release (67.86  0.73%). These findings confirmed that FUS improved the efficacy of BCNU release in-vitro.

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3.3. BCNU protection and prolonged half-life in circulation MBs not only transport drugs, but also protect and prolong the circulatory half-life of drugs, due to their pegylated surface modification (Fig. 6). The intact BBB of normal rat brains blocked any noticeable drug accumulation when BCNU was delivered as either BCNU-MB alone or free BCNU (0.20  0.09 mg and 4.22  0.23 mg). In BCNU group, bare BCNU hydrolyzed rapidly with short half-life of 16.3 min. However, in the BCNU-MB alone group, BCNU deposition slowly increased, reaching 2.98  0.38 mg at 30 min, suggesting that self-degradation of BCNU-MB at 37  C could result in the slow release of measurable amounts of BCNU. In the (MB þ FUS) þ BCNU group, FUS with MBs facilitated both local BBB opening and temporal local BCNU release (24.00  1.12 mg). However, BCNU was quickly hydrolyzed in the blood stream, leading to a significant drop in local BCNU concentration (1.93  0.25 mg) to undetectable levels at 30 min (half-life of 13.5 min) despite temporal opening of the BBB for > 1 h. When combining FUS with BCNU-MBs, BCNU also accumulated in the BBB-opened brain hemisphere (17.87  1.11 mg) at similar levels to (MB þ FUS) þ BCNU delivery. However, due to the selfprotecting nature of BCNU-MBs to prevent fast hydrolysis of BCNU in the blood stream and RES uptake, BCNU delivery to the brain remained high at 30 min (16.53  0.88 mg) and at > 50% after 60 min (10.85  0.96 mg). The long circulatory half-time of BCNUMBs (67.5 min) could thus allow delivery of more BCNU into the target brain during multiple FUS treatments with the same dose of injected BCNU.

3.2. In vivo FUS-induced BBB opening and BCNU release in normal rats Next we investigated whether the combination of BCNU-MBs with FUS could be used to both open the BBB and release BCNU. Fig. 5A demonstrates a typical case that employed 1-MHz FUS to disrupt the BBB of the hemispherical brain (acoustic pressure ¼ 0.5 MPa; burst length ¼ 5 ms; cycle number ¼ 5000; PRF ¼ 5 Hz; sonication duration ¼ 1 min/site; two sonicated sites). The BBBD region is stained with EB, and was sufficiently large to allow enhanced BCNU deposition over the whole area of the implant tumor. Optimal FUS exposure parameters for maximal BCNU release were determined. BCNU levels increased with acoustic pressure and reached a maximum at 0.5 MPa (17.87  1.11 mg), consistent with the in-vitro destruction data (Fig. 5B). However, further increasing the acoustic pressure caused a decrease to 12.08  2.33 mg BCNU. BCNU levels increased monotonically with PRF, but plateaued at 5 Hz (18.73  1.48 mg) (Fig. 5C). Multiple sonication sites were found to increase BCNU deposition, with a 1.5, 2.5, and 4-fold enhancement compared to each corresponding non-treated brain hemisphere (Fig. 5D). A total of 21.39  1.74 mg BCNU was released from 4-point sonicated brains. To evaluate treatment efficacy in the following experiments, FUS exposure was set to 0.5 MPa with pulse length 10 ms, 10000 cycle number, duty cycle 5%, and 2 min of sonication.

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Tumor progression was slower in the BCNU group, from 12.85  9.05 mm3 on day 10e56.78  38.80 mm3 on day 17 (4.8fold), but BCNU did not provide efficient control of tumor progression (150.83  11.48 mm3 on day 31). Median survival (29.5 days) was similar to the control group demonstrating limited efficacy of BCNU for glioma treatment. However, maximum animal survival improved to 43 days. Among the three experimental groups, only the BCNUMB þ FUS group demonstrated successful control of tumor progression. Tumor size decreased from 27.80  7.80 mm3 on day 10e13.40  4.12 mm3 on day 17 (0.5-fold), and the final measurement on day 31 was still well-controlled (11.0  1.0 mm3). The median survival was extended to 32.5 days (about a 12% increase compared to the BCNU and control groups), and the maximum animal survival was significantly increased to 59 days.

30 min–non-sonicated side 60 min–sonicated side 60 min–non-sonicated side

BCNU(ug)

25 20 15 10 5 0 BCNU-MB w/o FUS

BCNU-MB + FUS

BCNU

(MB+FUS) + BCNU

Fig. 6. Levels of drug accumulation in brain tissue for the indicated treatment groups at 10, 30 and 60 min after treatment. Single asterisk, p < 0.05; double asterisk, p < 0.01.

4. Discussion 4.1. Significance

3.4. BCNU liver accumulation and liver function evaluation

In this study, we demonstrated the successful use of smart, multifunctional MBs capable of BCNU-loading, FUS-BBBD induction, and local BCNU release for brain glioma treatment. We showed that BCNU can be encapsulated in the phospholipids of the MB shell with a high drug payload compared to other drug-loaded MB formulations [21,25]. Encapsulation of BCNU in MBs improved drug half-life in vivo (from 16.3 min to 1 h), and significantly reduced acute liver BCNU accumulation (approximately 5-fold) to reduce systemic BCNU cytotoxicity. In addition, we demonstrated that targeted BCNU delivery was significantly enhanced by BCNUMBs and FUS, profoundly improving its antitumor efficacy. Our study provides a new approach for designing multifunctional MBs to facilitate FUS-mediated chemotherapy for brain tumor treatment.

Since the IV-injected drugs are mainly metabolized through the liver, we evaluated their cytotoxic effects on liver (Fig. 7A). IVinjected BCNU rapidly accumulated to 113.57  3.62 mg in the liver. Liver accumulation decreased slightly (70.63  5.11 mg) in the (MB þ FUS) þ BCNU group, possibly due to the increased retention of BCNU in the brain. However, BCNU accumulation in the liver was significantly reduced when the drug was encapsulated in MBs. This reduction was independent of FUS brain sonication (12.22  3.24 mg without FUS, and 23.87  3.55 mg with FUS), translating to an accumulated liver dose of 9- and 5-fold less, respectively, in comparison to the BCNU-only group, and a potentially profound reduction in acute liver toxicity. We noted that both BCNU and BCNU-MB increased AST and ALT values (Fig. 7B and C), indicating possible liver damage. However, encapsulation of BCNU in MBs was associated with lower levels of AST or ALT compared to free BCNU, suggesting that BCNU-MBs may effectively reduce liver toxicity and damage.

4.2. Efficiency of BCNU release from MBs We found that the efficiency of BCNU-release plateaued at an acoustic pressure of 0.5 MPa, with about 70% measurable drug release. BCNU is efficiently hydrolyzed at increased temperatures and pH values, providing a possible explanation for the observed lack of complete BCNU release from MBs. Dilution of BCNU-MB in 0.9% normal saline to simulate in vivo conditions may also have cause hydrolysis, leading to underestimation of drug release. Another explanation may be diffusion of the MB gas core out of the lipid shell upon FUS exposure which may hamper the vibration or destruction of BCNU-MBs and hinder drug release. Finally, handling of samples before quantification (filtering, etc.) may have led to small losses.

3.5. Enhanced drug delivery and treatment efficacy in tumorimplanted rats Next we evaluated the combinatorial use of BCNU-MBs and FUS to enhance local drug delivery and improve treatment efficacy. Tumor size was measured by T2-weighted MRI (Fig. 8) and tumor volume was compared among the treatment groups (Fig. 9). Tumors progressed rapidly in the control animal group, from 22.07  17.57 mm3 on day 10e117.0  73.59 mm3 on day 17 (7.8fold), and finally to 202  24.10 mm3 on day 31. The median and maximal survival was 29 and 38 days, respectively.

A ∗∗

100 80 60 40 20



0 BCNU-MB BCNU-MB w/o FUS + FUS

BCNU

(MB+FUS) + BCNU

1600 1400 1200 1000 800 600 400 200 0

∗ ∗



120



100

∗∗

80 U/L

120

U/L

Liver BCNU (ug)

B ∗∗

140

∗∗

C ∗∗

∗∗

60 40 20 0

BCNU-MB BCNU-MB w/o FUS + FUS

BCNU

(MB+FUS) + BCNU

control

BCNU-MB BCNU-MB w/o FUS + FUS

BCNU

(MB+FUS) + BCNU

control

Fig. 7. (A) BCNU deposition in the liver 10 min after the indicated treatments. (B) AST (Aspartate aminotransferase) and (C) ALT (Alanine amino- transferase) measured at 24 h after the indicated treatments. Single asterisk, p < 0.05; double asterisk, p < 0.01.

C.-Y. Ting et al. / Biomaterials 33 (2012) 704e712

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Fig. 8. T2-weighted MRI to monitor brain tumor treatment outcomes at 10, 17, 24, and 31 days after tumor implantation. The corresponding 3D reconstructions of rat brains with tumors on day 31 are also presented. Animals were implanted with C6 glioma cells and received no treatment (control), treatment with BCNU on day 4 and 5 (BCNU), or treatment with BCNU-MBs following FUS irradiation on day 4 and 5 (BCNU-MB þ FUS). Tumor areas are delineated by yellow dotted lines. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

4.3. Influence of intracerebral hemorrhage on BCNU release Excessive FUS pressure is known to cause intracerebral hemorrhage (ICH) in the exposed brain region [20]. Liu et al. found that ICH occurred at pressures of 0.98 MPa or higher, with a simultaneous reduction in BCNU delivery [27]. ICH may affect both the diffusion of BCNU from the microvasculature into brain tissues, and its hydrolysis, resulting in underestimation of the effective BCNU concentration [25,27]. It is therefore crucial to use optimal sonication parameters to enhance release of BCNU from BCNU-MBs while preventing ICH.

confirmed that PEG on BCNU-MBs can effectively reduce RES/ phagocytes uptake to delay the immune-reaction and reduce drug accumulation in the liver. Nevertheless, BCNU-MBs are eventually cleared by the RES, so some long-term damage of metabolizing organs may still occur. We found that the BCNU-MB þ FUS system induced less liver function depression than other ways of drug delivery. This suggests that encapsulation of BCNU in MB carriers reduced drug exposure and injuries to other normal tissues. However, the initial lower dose of BCNU in the blood for the BCNUMB þ FUS groups compared to the BCNU and (MB þ FUS) þ BCNU groups may also explain the decrease in damage to liver function since less of the free drug circulated through the liver.

4.4. Implications of the self-protective nature of BCNU-MBs 4.5. Off-target accumulation of BCNU released from MBs

Normalized tumor volume (%)

We found that free BCNU was easily hydrolyzed in the plasma, ultimately reducing treatment efficacy. However, when protected by MB carrier, BCNU remained in circulation for much longer for delivery to brain tissues. Surface modification of liposomes with PEG is known to not only reduce acute toxicity but also delay side effects and initiate self-repair of macrophages [31]. Here we

25

Control BCNU-MB+FUS

20

BCNU

15 10

4.6. In vivo antitumor efficacy

5 0

In the non-sonicated (right) hemisphere of the brain, the deposition of BCNU in the (MB þ FUS) þ BCNU group was two-fold higher than in the BCNU-MB þ FUS group (9.20  1.18 mg and 5.35  0.36 mg, respectively). Since the destruction of BCNU-MBs by FUS was not complete, the exposure of chemotherapeutic drugs to the blood was lower in the BCNU-MB þ FUS group than in the (MB þ FUS) þ BCNU group. However, delivery of BCNU to the nonsonicated brain hemisphere was higher for both these groups compared to the free BCNU group. This could be attributed to the wide beam width (about 2 mm) of the 1-MHz FUS probe, which may have irradiated part of the right hemisphere causing a small amount of BBB opening and some drug deposition.

0

10

20

30

40

days Fig. 9. (A) Tumor volume analysis from MR images. (A) Control group, (-) BCNUMB þ FUS group, and (C) BCNU group. Double asterisks, p < 0.01.

Pre-clinical and clinical trials have shown that combining drugs with enhanced BBB permeability can improve treatment outcomes [18,27,32]. The method of drug delivery proposed here has the advantage of both enhancing the permeability of the BBB, and the efficacy of local chemotherapy. Our results clearly indicated that the use of BCNU-MB with FUS to improve delivery of BCNU into the brain tumors had the best effect on the suppression of tumor progression.

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5. Conclusions BCNU was successfully encapsulated in MB shells leading to enhancement of drug half-life in vivo and controlled drug release into brain tissue by FUS sonication. BCNU-MBs used in combination with FUS provide a promising strategy to transport chemotherapeutic drugs across the BBB and enhance the local drug concentration in targeted brain locations while reducing systemic cytotoxic effects. Acknowledgements The authors acknowledge the supports of National Science Council of Taiwan (NSC 99-2218-E-182-002 and 98-2320-B-007002-MY3), and Professors R.-A. Doong and Y.-F. Huang for their help with experiments. References [1] Louis DN, Ohgaki H, Wiestler OD, Cavenee WK, Burger PC, Jouvet A, et al. The 2007 WHO classification of tumours of the central nervous system. Acta Neuropathol 2007;114:97e109. [2] Arko L, Katsyv I, Park GE, Luan WP, Park JK. Experimental approaches for the treatment of malignant gliomas. Pharmacol Ther 2010;128:1e36. [3] Huse JT, Holland EC. Targeting brain cancer: advances in the molecular pathology of malignant glioma and medulloblastoma. Nat Rev Cancer 2010; 10:319e31. [4] Jemal A, Siegel R, Ward E, Murray T, Xu J, Smigal C, et al. Cancer statistics. CA Cancer J Clin 2006;56:106e30. [5] Kang C, Yuan X, Zhong Y, Pu P, Guo Y, Albadany A, et al. Growth inhibition against intracranial C6 glioma cells by stereotactic delivery of BCNU by controlled release from poly(D, L-lactic acid) nanoparticles. Technol Cancer Res Treat 2009;8:61e70. [6] Chen PY, Liu HL, Hua MY, Yang HW, Huang CY, Chu PC, et al. Novel magnetic/ ultrasound focusing system enhances nanoparticle drug delivery for glioma treatment. Neuro Oncol 2010;12:1050e60. [7] Westphal M, Hilt DC, Bortey E, Delavault P, Olivares R, Warnke PC, et al. A phase 3 trial of local chemotherapy with biodegradable carmustine (BCNU) wafers (Gliadel wafers) in patients with primary malignant glioma. Neuro Oncol 2003;5:79e88. [8] Marosi C. Chemotherapy for malignant gliomas. Wien Med Wochenschr 2006; 156:346e50. [9] Vogelhuber W, Spruss T, Bernhardt G, Buschauer A, Göpferich A. Efficacy of BCNU and paclitaxel loaded subcutaneous implants in the interstitial chemotherapy of U-87 MG human glioblastoma xenografts. Int J Pharm 2002; 238:111e21. [10] Engelhard HH. The role of interstitial BCNU chemotherapy in the treatment of malignant glioma. Surg Neurol 2000;53:458e64. [11] Li Y, Ho Duc HL, Tyler B, Williams T, Tupper M, Langer R, et al. In vivo delivery of BCNU from a MEMS device to a tumor model. J Control Release 2005;106: 138e45. [12] Khil MS, Kolozsvary A, Apple M, Kim JH. Increased tumor cures using combined radiosurgery and BCNU in the treatment of 9l glioma in the rat brain. Int J Radiat Oncol Biol Phys 2000;47:511e6.

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