Augmented Blood-Tumor Barrier Permeability and Enhanced Doxorubicin Delivery in Rat Brain Tumor Models Through Additional Focused Ultrasound Stimulation

Augmented Blood-Tumor Barrier Permeability and Enhanced Doxorubicin Delivery in Rat Brain Tumor Models Through Additional Focused Ultrasound Stimulation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Augmented Blood-Tumor Barrier Permeability and Enhanced Doxorubicin Delivery in Rat Brain Tumor Models Through Additional Focused Ultrasound Stimulation Hyo Jin Choi, Mun Han, Byeongjin Jung, Hyungkyu Huh, Eun-hee Lee, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5202132/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 24 Feb, 2025 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Focused ultrasound (FUS) has emerged as a promising technique for temporarily disrupting the blood-brain barrier (BBB) and blood-tumor barrier (BTB) to enhance the delivery of therapeutic agents. Despite its potential, optimizing FUS to maximize drug delivery while minimizing adverse effects remains a significant challenge. In this study, we evaluated a novel FUS protocol that incorporates additional FUS stimulation without microbubbles (MBs) ("FUS protocol") prior to conventional BBB disruption with MBs ("BBBD protocol") in a rat brain tumor model (n = 35). This approach aimed to validate its effectiveness in enhancing BBB/BTB disruption and facilitating doxorubicin delivery. T1-weighted contrast-enhanced and dynamic contrast-enhanced (DCE) MRI demonstrated significant increases in signal intensity and permeability (K trans ) in the tumor region under the "FUS + BBBD protocol”, with 2.65-fold and 2.08-fold increases, respectively, compared to the non-sonicated contralateral region. These values were also elevated compared to the conventional "BBBD protocol" by 1.45-fold and 1.25-fold, respectively. Furthermore, doxorubicin delivery in the targeted region increased by 1.91-fold under the "FUS + BBBD protocol”, compared to a 1.44-fold increase using the conventional "BBBD protocol”. This novel FUS approach offers a promising, cost-effective strategy for enhancing drug delivery to brain tumors. While further studies are required to assess its applicability with different chemotherapeutics and tumor types, it holds significant potential for improving brain tumor treatment in both preclinical and clinical settings. Biological sciences/Cancer Biological sciences/Neuroscience Physical sciences/Energy science and technology Focused ultrasound Blood-tumor barrier Mechanical stress Doxorubicin Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction The blood-brain barrier (BBB) is an obstacle to the effective treatment of brain tumors because it limits drug delivery and penetration 1 , 2 . Vigorous angiogenesis in tumors forms a heterogeneous vasculature with a compromised BBB, the so-called blood-tumor barrier (BTB) 3 . Due to its permeability, the BTB allows for the extravasation of small and large molecules 4 – 7 . However, the BTB permeability is highly heterogeneous depending on the type, stage, and lesion of the brain tumor. Hence, the controlled delivery of adequate amounts of therapeutic agents for tumor treatment is still unclear 7 , 8 . Radiation therapy combined with chemotherapy is currently used as the international standard of care for brain tumor treatment. Alternative methods, such as mannitol and bradykinin, have been attempted to increase the amount of therapeutic agent delivery for effective tumor treatment 9 . However, limitations such as unpredictable BBB disruption can pose a potential risk to normal brain tissue 10 . Various experimental approaches, including the use of viral vectors 11 , polymeric nanoparticles 12 , and exosomes 13 , have also been investigated to improve drug delivery across the BBB and BTB (hereafter referred to as BBB/BTB) 14 . Unfortunately, these methods are not yet used in clinical practice owing to some limitations such as difficulties in manufacturing, high production cost, safety 15 , 16 and therapeutic efficacy due to low accumulation 17 . Focused ultrasound (FUS) combined with intravenously injected microbubbles (MBs) has recently emerged as a promising non-invasive approach for disrupting the BBB/BTB. Preclinical studies using glioma models in animals have demonstrated its potential to enhance the delivery of therapeutic agents, including trastuzumab 18 , doxorubicin 19 , temozolomide 20 , methotrexate 21 , and carboplatin 22 , to targeted brain tumor regions. These studies have shown tumor suppression and improved survival rates in rat and mouse brain tumor models. Additionally, several clinical studies have confirmed the safety and feasibility of focused ultrasound BBB disruption (FUS-BBBD) using various devices, multiple treatment sessions, and various chemotherapies in patients with newly diagnosed GBM, recurrent GBM, and high-grade gliomas 23 . Despite the progress made in preclinical and clinical trials, further optimization of FUS parameters, including exposure settings and MB doses, is needed to minimize the risk of hemorrhage and other adverse effects while maximizing therapeutic outcomes 24 – 26 . We previously reported that an additional FUS stimulation (“FUS protocol”) step without MB injection prior to the conventional BBBD (“BBBD protocol”) enhances BBB permeability and doxorubicin (DOX) delivery to the brain of healthy rats without damaging nearby tissues or vessels 27 . However, the robustness of this new FUS protocol (“FUS + BBBD protocol”) has not been validated in the tumor microenvironment. As a follow-up study, we tested the efficacy and efficiency of the FUS-BBBD protocol in the tumor rat model. Methods Animals and husbandry All experiments were approved by the Animal Experiment Ethics Committee of Daegu–Gyeongbuk Medical Innovation Foundation (IACUC No. DGMIF-20122901-00). All procedures were conducted in accordance with the relevant guidelines. The study was also conducted in compliance with the ARRIVE (Animal Research: reporting of in vivo experiments) guidelines ( https://arriveguidelines.org/arrive-guidelines ). All efforts were made to minimize the number of animals and their pain. Male Sprague-Dawley rats (5 weeks old, weighing 100–150 g for tumor drug delivery study, n = 35) were purchased from Orient Bio Inc. (Sungnam, Republic of Korea). All rats were used after 7 days of acclimatization in the vivarium of the Laboratory Animal Center (LAC) of the Daegu-Gyeongbuk Medical Innovation Foundation (DGMIF). Rats were fed a normal diet with water during acclimatization. The temperature (20–25°C) and humidity (40–45%) were controlled, and a 12:12 h light/dark cycle in polycarbonate cages. The rats used in this study were randomly divided into five groups based on the sonication protocol (Table 1 ). Table 1 Summary of the sonication protocol groups. Experimental purpose Sonication protocol No of sonication targets Tumor model BBB permeablization (T1 and DCE-MRI Ctrl 12 1.0F 4 B 4 1.0FB 4 Drug delivery Ctrl 12 1.0F * 5 B ** 8 0.5FB *** 5 1.0FB **** 6 2.0FB ***** 8 Histopathology 1.0F 3 B 1.0FB Normal Immunostaining 1.0F 6 B 1.0FB * 1.0F: 1.0MPa w/o MB; ** B: 0.72MPa w/ MB; *** 0.5FB: 0.5MPa w/o MB + 0.72MPa w/ MB; **** 1.0FB: 1.0MPa w/o MB + 0.72MPa w/ MB; ***** 2.0FB: 2.0MPa w/o MB + 0.72MPa w/ MB Reagents Doxorubicin hydrochloride (DOX) (Boryung Pharmaceutical, Seoul, Republic of Korea) was used to confirm drug delivery in the tumor environment. Evans blue (Sigma-Aldrich St. Louis, MO, USA) was used to demonstrate the BBB opening region. Zoletil (Virbac Laboratories, Carros, France) and Rompun (Bayer, Leverkusen, Germany) were used for animal anesthesia. Microbubbles (MBs) Definity® were purchased (Lantheus Medical Imaging, North Billerica, MA, USA) for BBB opening along with FUS, and gadoterate meglumine (Gd-DOTA) was purchased (Dotarem ® ; Guerbet, Roissy CDG, France) to identify the BBB opening region using MRI. 9L gliosarcoma cell culture 9L gliosarcoma cell lines (ATCC® CRL-2200 ™ ) were cultured at 37°C and 5% CO 2 in Dulbecco Modified Eagle Medium (DMEM; 11995-073, Gibco, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (FBS; 16000-044, Gibco, Gaithersburg, MD, USA) and 1% penicillin-streptomycin (PS; 15140-122, Gibco, Gaithersburg, MD, USA), as previously described 20 , 28 , 29 . At approximately 90% cell confluence, the media was removed and the cells were removed using 0.25% trypsin-EDTA (25200-056, Gibco, Gaithersburg, MD, USA). To prepare the tumor cells for implantation, they were centrifuged at 1,000 rpm for 2 min, suspended in DMEM at a final concentration of 5×10 5 cells/5 µL, and kept cool until implantation. Before tumor implantation, the 9L gliosarcoma stock was thawed and used for the experiment after the acclimatization period. All brain tumor implantations were carried out with 9L gliosarcoma at passage number 5–7. Brain tumor implantation procedure The rats were anesthetized with an intramuscular injected anesthetic mixture [Zoletil (25 mg/kg): Rompun (4.6 mg/kg): saline = 5:2:3] prior to tumor implantation. The rat was fixed to the stereotaxic frame (Harvard Apparatus, Holliston, MA, USA) using an in-ear and upper tooth bar for hemostasis. Animal hair was shaved, and a sagittal incision was made on the posterior aspect of the head, approximately 2 cm. A small burr hole was drilled using a dental drill (Saeshin, Daegu, Korea) 3 mm lateral and 1 mm anterior to the bregma . The 9L gliosarcoma cells filled in a 25 µl Hamilton syringe (Hamilton Company, Reno, NV, USA) were slowly injected at 5 mm under the dura mater at a flow rate of 1 µL/min for 5 min using a 30 gauge Hamilton needle (Hamilton Company, Reno, NV, USA) and a syringe pump (Harvard Apparatus, Holliston, MA, USA). To avoid any cell leakage or backflow of the media, holes were filled with Hy-bond polycarboxylate cement (Shofu, TYOKO, Japan) and the skin was sutured. After 10–14 days of implantation, tumor size was checked using a horizontal bore 9.4T animal MRI (BioSpec 94/20 USR; Bruker, Billerica, MA, USA) for controlled experiments. Total 35 rats that presented with a tumor diameter of 1.5 ~ 2.0 mm based on T2-weighted (T2W) coronal images were selected and used in the experiment. MRI-guided Focused Ultrasound (MRgFUS) system The MRgFUS system (RK-100; FUS Instruments, Toronto, Canada) was used to sonicate rat brains for the BBB as described previously 27 , 30 . A schematic of the system is presented in Fig. 1 . Briefly, the system has an air-back, single-element, and spherical piezoelectric transducer (FUS Instruments, Toronto, Canada). A spherical piezoelectric transducer with a diameter of 75 mm and a radius of curvature of 60 mm, which generates a resonant frequency of 1 MHz, was used. The ultrasound − 3 dB acoustic pressure width and length of the focal region were 1.5 mm and 6 mm in free water at the peak pressure measured by the Acoustic Intensity Measurement System (AIMS III, ONDA, Sunnyvale, CA, USA) and the hydrophone (HGL-400, ONDA, Sunnyvale, CA, USA) 30 . The transducer was driven by a waveform generator (33220A; Agilent, Santa Clara, CA, USA) and an RF power amplifier (4010L; E&I, Rochester, NY, USA). The transducer was submerged in a degassed water tank. A 9.4T MRI and a volume coil with an 86-mm inner diameter were used for image guidance by syncing the coordinates between the two systems (MRI and MRgFUS system). Sonication protocol Sonication was performed using the MRgFUS system, as described in section 2.5. Burst sonication (10ms tone burst, pulse repetition frequency (PRF) of 1Hz for 120 s) was performed at the location of the tumor. The acoustic pressure of the FUS protocol “F” was 0.5, 1.0, and 2.0 MPa depending on the experimental purpose (Table 1 ) and the acoustic pressure of the BBBD protocol “B” was 0.72 MPa. The FUS + BBBD protocol “FB” was a fusion of both, where “FUS protocol” ultrasound was applied prior to the “BBBD protocol” without MB injection. The details of the experimental schedule for the sonication protocols are presented in Fig. 2 . These sonication parameters have been verified as safe conditions in previous studies 27 , 30 , 31 . Although BBB/BTB is simultaneously disrupted in the tumor animal model, protocol used in this study is referred as BBBD throughout the manuscript in order to minimize confusion and to be consistent with previous studies. Magnetic resonance imaging A 9.4T preclinical animal MRI was used for magnetic resonance imaging. T1-weighted (T1W) MR images were used to check the BBB disruption. T2-weighted (T2W) MR images were used as a guide for ultrasound targeting and tumor size checking. Meanwhile, DCE-MRI was used to identify the permeability 19 , 27 , 30 . MRI parameters were as follows: T1W, echo time (TE) = 6.5 ms, repetition time (TR) = 1500 ms, field of view (FOV) of 40 × 40 mm 2 , matrix size of 256 × 256, axial and coronal slices of 1.5 mm slice thickness without a gap, and the number of excitations (NEX) = 3. T2W: TE = 33 ms, TR = 2500 ms, FOV = 40 × 40 mm 2 , matrix size = 256 × 256, axial and coronal slices of 1.5 mm slice thickness without a gap, and NEX = 2. DCE-MRI: TE = 1.5 ms, TR = 24.3 ms, FOV = 40 × 40 mm 2 , matrix size of 128 × 128, axial and coronal slices of 1.5 mm slice thickness without a gap, and NEX = 3. The MRI parameters are summarized in Table. 2. Table 2 MR Imaging Parameters. Sequence Use TE * (ms) TR ** (ms) FOV *** (mm 2 ) Matrix Size Slice Thickness (mm) NEX **** RARE T1-weighted Detection of BBB disruption 6.5 1500 40×40 256×256 1.5 3 RARE T2-weighted Sonication target planning/ Detection of edema/ Tumor size checking 33 2500 40×40 256×256 1.5 2 FLASH DCE-MRI Permeability measurement 1.5 24.3 40×40 128×128 1.5 3 * TE: Echo time; ** TR: Repetition time; *** FOV: Field of view; **** NEX: Number of excitations The region of interest (ROI), which is the focal area of the transducer, was selected as a circle with a diameter of 1.5 mm. The relative signal intensity R t of the tumor region at a given time point (T1 t ) was normalized by the signal intensity of pre-injection T1W MR images (0 min, T1 pre ) using the following equation: $$\:{R}_{t}\left(\text{\%}\right)=\:\frac{\left({T1}_{t}-{T1}_{pre}\right)}{{T1}_{pre}}\text{*}100$$ The BBB permeability K trans was calculated based on a Patlak model using DCE-MRI images, similar to a previous study 32 . T1 measurements were acquired using (RARE VTR) (TR = 160, 180, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1500, 1800, 2000, 2500, 3000, 3500, 4000, 6000, and 12000 ms). Ten sets of FLASH images were acquired before the contrast agent injection (Gd-DOTA with relaxivity of 3.0 s − 1 mM − ), followed by 90 sets of FLASH images acquired during and after CA injection with a temporal resolution of 9.32 s for 10 min. The same slice position was used for T1 mapping and DCE-MRI. The ROI was selected using a 5-pixel circle diameter, which corresponds to a 1.5 mm focal area of the transducer (3.2 pixels/mm). The plasma concentration of the contrast agent was measured in a large blood vessel (ophthalmic artery or transverse sinus). The hematocrit level was assumed to be 45% in the capillary 33 . Blood-brain barrier disruption experiment The blood-brain barrier and blood-tumor barrier disruption experiments were performed as described in our previous studies 27 , 30 . Before sonication, the tumor size was checked with T1-weighted MR images 10–14 days after tumor implantation. A tumor diameter of 1.5 ~ 2.0 mm was selected and used in this study. Briefly, rats were anesthetized with an intramuscular injection of an anesthetic mixture (Zoletil (25 mg/kg):Rompun (4.6 mg/kg): saline = 5:2:3). After anesthesia, an angiocatheter was inserted into the vein of the tail, and the hair on the head was shaved. Then, the rat was placed in a supine position on an MR-compatible plastic bed, and the exposed scalp was immersed in a degassed water bag using fixed ear and tooth bars (Fig. 1 ). Sonication of the tumor region was performed according to the protocol described in Fig. 2 . The T2W MR image was scanned and registered to the MRgFUS system and placed in sync. The 0.02 mL/kg perflutren lipid MB (Definity®) were diluted 1:50 in saline and administered through an angiocatheter using an automated syringe pump (Harvard Apparatus, Holliston, MA, USA). Sonication was then started 10 s after the diluted MB solution infusion. Following sonication, T1W MR or DCE-MR images were performed to identify BBBD and permeability with Gd-DOTA (0.2 mM/kg). DOX (5.67 mg/kg) and Evans blue (4 mg/kg) were administered intravenously (i.v.) to mark the DOX delivered region for tissue extraction. Doxorubicin quantification All rats were sacrificed 24 h after the IV administration of DOX and perfused with 0.9% NaCl via transcardial perfusion followed by brain extraction. Then, the sample volume (approximately 30 mg of tissue) of the tumor region was harvested for quantification. The tumor samples were homogenized with acidified ethanol (50% ethanol in 0.3 N HCl) using a bead beater and incubated for 24 h at 4°C. After incubation, the samples were centrifuged at 16,000 × g for 20 min to remove the tissue pellet, and the supernatant was collected. The fluorescence intensity was measured using a fluorometric detector with 480 nm excitation and 590 nm emission (Infinite 200 Pro; TECAN, Austria GmbH, Austria). The concentration of DOX was quantified using a standard curve derived from eight serial concentrations of pure DOX (0, 10, 20, 50, 100, 200, 500, and 1000 ng/mL). Fluorescence intensity was analyzed using TECAN i-control software (TECAN, Austria GmbH, Austria), and all measurements were repeated at least three times. The contralateral tumor region without sonication was used as a control to correct autofluorescence. DOX fluorescence detection and analysis were performed as described previously 27 , 31 , 34 . Hematoxylin and Eosin (H&E) staining Histopathological changes in the sonicated rat brain tissue were investigated by H&E staining as described previously 19 , 30 , 32 . The sonicated rats were sacrificed 24 hours post-sonication, followed by transcardiac perfusion with 0.9% NaCl and fixation with 10% formalin. The rat brains were extracted and fixed in 10% formalin for 7 days. The brain tissue samples were then embedded in paraffin and cut serially in 4 µm thickness axial direction using a microtome (HistoCore AUTOCUT; Leica, Wetzlar, Germany). Paraffin-embedded sections were deparaffinized and rehydrated before staining. Brain tissue slides were stained with an H&E stain kit (Vector Laboratories, Burlingame, CA, USA). Images were taken with a digital slide scanner (Axio Scan.Z1; Carl Zeiss, Oberkochen, Germany). Statistical Analysis The values obtained were expressed as mean ± standard deviation. Statistical significance differences between the control group and experimental groups were compared using a two-tailed paired t-test, one-way ANOVA with Tukey’s test, and two-way ANOVA with Tukey’s test using GraphPad Prism 8 (GraphPad Software, Inc., San Diego, CA, USA). Statistical significance was accepted for p -values < 0.05. Results BBB/BTB permeability To investigate the effect of the additional FUS stimulation on the BBB/BTB permeability, contrast-enhanced T1W MR imaging was performed for “B”, “1.0FB”, and “1.0F” as shown in Fig. 3 . Representative MR Images of each case show the average signal intensity of the contrast agent after BBB/BTB disruption (Fig. 3 A). For every given case, averaged signal intensity of the sonicated tumor region was higher than the non-sonicated contralateral “C” region in both “B” and “1.0FB” cases. However, this difference was not observed for the “1.0F” case. To quantify the BBB/BTB permeability, the relative signal intensity of the contrast agent was measured in a time-dependent manner (before, 0, 3, 5, 7, 9, and 11 min), as shown in Fig. 3 B. The relative signal intensity of the target area increased by 33.2 ± 3.1%, 41.2 ± 3.1%, 43.9 ± 3.3%, 45.9 ± 3.0%, 44.9 ± 2.7% and 55.0 ± 4.9%, 54.5 ± 3.4%, 51.1 ± 1.3%, 48.9 ± 0.7%, 47.9 ± 0.1% for each time point compared to the contralateral region under “B” and “1.0FB” protocol, respectively. However, the “1.0F” region showed no significant change compared to the contralateral “C” region. In these results, the relative signal intensity was highest (2.65 times compared to the contralateral region) at the “1.0FB” region, 3 min after the MR contrast agent injection. In addition to the averaged signal intensity changes, brain permeability (K trans ) was calculated using DCE-MRI for each protocol (“B”, “1.0FB”, and “1.0F”). The representative K trans map and group-averaged K trans values before and after the sonication procedure are shown in Figs. 4 A and 4 B. The average permeability of the tumor region (n = 4) sonicated by “B” and “1.0FB” protocol was enhanced by 0.0741 ± 0.0159 min -1 and 0.0928 ± 0.0186 min - 1 , respectively, which was 2.08-fold and 1.73-fold higher than that before sonication (Fig. 4 C). The increase in K trans after sonication was statistically significant (p = 0.031 and 0.0006 for “B” and “1.0FB” protocols, respectively. For the “1.0F” protocol, however, no significant increase in the brain permeability was observed (0.0379 ± 0.00701 min - 1 after sonication, compared to 0.0378 ± 0.00738 min - 1 before sonication with p = 0.99). DOX delivery The targeted brain tissue samples were measured for statistical comparison for DOX delivery for each protocol “B”, “1.0FB”, and “1.0F” as shown in Fig. 5 . The average DOX concentration was 694.1 ± 398.5 ng/g in the contralateral “C” region, while it was 885.1 ± 305.6 ng/g in the tumor region sonicated with “B”, 996.2 ± 408.0 ng/g for “0.5FB”, 1255.4 ± 347.8 ng/g for “1.0FB”, 1327.8 ± 470.7 ng/g for “2.0FB”, and 757.3 ± 447.4 ng/g for “1.0F”. The amount of DOX delivered under the “B”, “0.5FB”, “1.0FB”, “2.0FB”, and “1.0F” protocols was 1.28 ± 0.44, 1.44 ± 0.59, 1.81 ± 0.50, 1.91 ± 0.68, and 1.09 ± 0.64 times higher, respectively, than in the contralateral region without sonication “C”. Histopathological analysis H&E histopathological analysis (n = 3) was performed to identify the damage caused by “B”, “1.0FB”, and “1.0F” in the brain tumor region. No significant differences between regions sonicated with each protocol were observed in the 5-and 20 times magnified images, as shown in Fig. 6 . Discussion There are increasing clinical demands 35 – 37 on safe and efficient drug delivery techniques for tumor treatment. A temporal BBB/BTB opening using focused ultrasound was suggested as a possible candidate 38 – 40 . Previous research demonstrated that incorporating an additional sonication step prior to conventional BBBD can enhance drug delivery in healthy rat brains by up to 1.75 times, without inducing tissue damage or heating 27 . This additional sonication step, performed without microbubble injection, has shown to be a simple yet effective means of increasing drug delivery. The present study focuses on validating this protocol in tumor models, aiming to support its potential translation into clinical applications. Consistent with previous experiments in normal rats, the addition of ultrasound sonication significantly enhanced BBB/BTB opening in the tumor environment. Given that the BTB is known to be more permeable than the intact blood-brain barrier 4 – 7 , baseline BTB permeability was assessed through MR imaging 24 hours prior to sonication to minimize experimental bias. Using the “1.0FB” protocol, the signal intensity of T1-weighted MR and K trans increased by approximately 2.42-fold and 2.08-fold, respectively, compared to pre-sonication measurements at the same target, and by 2.65-fold and 2.15-fold relative to the contralateral tumor region. Compared to the conventional BBBD protocol “B”, the addition of ultrasound stimulation resulted in a 1.45-fold increase in BTB permeability. This is consistent with previous findings, where additional sonication prior to BBBD increased the average Ktrans in normal tissue by 1.36-fold compared to the conventional BBBD protocol 27 . Despite differences in the permeability characteristics of the BBB/BTB between the current tumor model and normal brain tissue, the addition of an extra sonication step prior to BBBD consistently produced a comparable increase in permeability. The amount of chemotherapeutic agent delivered was also compared after sonication using each protocol (Fig. 5 ). In order to minimize experimental bias, animals with a tumor size of less than 20% difference were selected and used (and tumor diameter between 1.5 ~ 2.0 mm). The delivery of DOX to the target tumor region sonicated using the “1.0FB” protocol was 1.44-fold higher than that achieved with the conventional BBBD protocol “B”. The observed patterns in permeability and drug delivery were similar to those reported in a previous study 27 , although the differences were not statistically significant. It is important to note that the previous study involved opening an intact BBB in a normal animal model, while the BTB in the brain tumor model used in this study was already more permeable and heterogeneous 41 , 42 . Additionally, the untreated contralateral brain tumor region exhibited variability in integrity due to existing BBB dysfunction. Both contrast agents and DOX showed heterogeneous permeability, even in the absence of ultrasound, which likely influenced the statistical significance of the findings 43 – 45 . An additional sonication step prior to BBBD was shown to enhance BBB/BTB permeability, thereby increasing the overall delivery of chemotherapeutic agents in the tumor model. To assess the safety of this new protocol in the brain tumor environment, histopathological examination of brain tissue was performed using standard H&E staining to evaluate potential brain damage 46 , 47 . As shown in Fig. 6 no significant differences were observed in the “1.0FB” group compared to the “B” and “1.0F” groups. The enhancement of brain permeability by adding an extra sonication step prior to conventional BBBD (ultrasound with intravenously injected microbubbles) may increase sensitivity to acoustic energy by affecting the cytoskeleton and extracellular matrix through dynamic remodeling and matrix degradation 48 . Another possibility is that the increased permeability results from the expansion of extracellular and perivascular spaces, rather than changes in the BBB itself. Recent studies have highlighted the role of mechanical stress on mechanosensitive calcium ion channels 49 – 52 . Among these, TRP channels are widely expressed and activated by various stimuli, with the mammalian TRP channel family divided into six subfamilies: canonical (TRPC1-7), vanilloid (TRPV1-6), melastatin (TRPM1-8), polycystin (TRPP1-5), mucolipin (TRPML1-3), and ankyrin (TRPA1) 53 – 55 . These ion channels are known to compromise BBB integrity, suggesting that prior FUS stimulation may enhance BBB disruption, potentially explaining the current findings 51 . Further studies are required to fully understand the underlying mechanism of the drug delivery enhancement. DOX is widely used in chemotherapeutic drug delivery studies. However, other therapeutic agents that could benefit from enhanced drug delivery were not evaluated in this study. For instance, TMZ is the primary treatment option for gliomas. It is a relatively small lipophilic molecule that can cross the BBB 56 . It has been shown that TMZ levels in the brain and cerebrospinal fluid reach up to 20% of the drug plasma concentration 57 . However, the therapeutic potential of TMZ for glioma treatment is limited by its short half-life (1.8 h). This requires continuous drug administration to maintain the therapeutic concentration of the drug in tumor tissue and to optimize therapeutic potential 58 . The proposed FUS + BBBD protocol may offer a safe and effective method for delivering TMZ in the near future. In addition, only a well-characterized 9L gliosarcoma that is both highly immunogenic and poorly infiltrative was used in this study 28 , 59 , 60 . Future studies should include a broader range of brain tumor models, such as glioma, glioblastoma, medulloblastoma, and brain metastasis, as each tumor type may present different characteristics due to tumor heterogeneity 24 . Finally, further study is required to validate the effect of mechanosensitive on calcium ion channels 51 , 52 . Concluding remarks In conclusion, the additional mechanical stress from ultrasound stimulation prior to conventional BBBD improves overall permeability in the target tumor region, leading to enhanced drug delivery. The proposed advanced “FUS + BBBD protocol” represents a promising strategy to increase the delivery of chemotherapeutic agents with minimal side effects in brain tumor treatment. Further research is necessary to elucidate the biomolecular and cellular mechanisms underlying the improved BBBD achieved with this protocol, both in preclinical studies and potential clinical applications. Declarations Acknowledgements This research was supported by the Korea Health Technology R&D Project through the Korea Health Industry Development Institute, funded by the Ministry of Health & Welfare, Republic of Korea (Grant number: RS-2023-KH135060, and RS-2024-00337463). Also, this research was support by the Starting growth Technological R&D Program, funded by the Ministry of SMEs and Startups, Republic of Korea (Grant number: RS-2023-00261874). In addition, we would like to appreciate Yuri Hong who supported biological assays in this study. Author contributions Hyo Jin Choi: Methodology, Histopathological sample preparation, Analysis and validation, Writing—draft preparation, Writing—review and editing Mun Han: Methodology, In vivo preclinical experiment, Analysis and validation, Writing—draft preparation, Writing—review and editing Byeongjin Jung: Methodology, In vivo preclinical experiment, Writing—draft preparation Hyungkyu Huh: Analysis and validation, Writing—draft preparation Eun-hee Lee: Analysis and validation, Writing—review and editing Jong-ryul Choi: Analysis and validation, Writing—review and editing Juyoung Park: Supervision, Conceptualization, Funding acquisition, Methodology, Analysis and validation, Writing—draft preparation, Writing—review and editing. Competing interests The authors declare no conflict of interest. Data statement All data generated or analyzed during this study are included in this published article. 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M. & McDannold, N. Ultrasound-mediated blood-brain barrier disruption for targeted drug delivery in the central nervous system. Adv. Drug Deliv. Rev. 72 , 94–109. 10.1016/j.addr.2014.01.008 (2014). Hynynen, K., McDannold, N., Vykhodtseva, N. & Jolesz, F. A. Noninvasive MR imaging-guided focal opening of the blood-brain barrier in rabbits. Radiology . 220 , 640–646. 10.1148/radiol.2202001804 (2001). Meairs, S. Facilitation of Drug Transport across the Blood-Brain Barrier with Ultrasound and Microbubbles. Pharmaceutics . 7 , 275–293. 10.3390/pharmaceutics7030275 (2015). Stewart, D. C., Rubiano, A., Dyson, K. & Simmons, C. S. Mechanical characterization of human brain tumors from patients and comparison to potential surgical phantoms. PloS one . 12 , e0177561. 10.1371/journal.pone.0177561 (2017). Mancia, L. et al. Focused ultrasound ablation of solid tumors: Feasibility of planning tissue-selective treatments. J. Clin. Oncol. 38 , e15600–e15600. 10.1200/JCO.2020.38.15_suppl.e15600 (2020). O'Brown, N. M., Pfau, S. J. & Gu, C. Bridging barriers: a comparative look at the blood-brain barrier across organisms. Genes Dev. 32 , 466–478. 10.1101/gad.309823.117 (2018). Abbott, N. J. Blood-brain barrier structure and function and the challenges for CNS drug delivery. J. Inherit. Metab. Dis. 36 , 437–449. 10.1007/s10545-013-9608-0 (2013). Ewing, J. R. et al. Model selection in magnetic resonance imaging measurements of vascular permeability: Gadomer in a 9L model of rat cerebral tumor. Journal of cerebral blood flow and metabolism. official J. Int. Soc. Cereb. Blood Flow. Metabolism . 26 , 310–320. 10.1038/sj.jcbfm.9600189 (2006). Li, Y. et al. Hematoxylin and eosin staining of intact tissues via delipidation and ultrasound. Sci. Rep. 8 , 12259. 10.1038/s41598-018-30755-5 (2018). Chan, J. K. The wonderful colors of the hematoxylin-eosin stain in diagnostic surgical pathology. Int. J. Surg. Pathol. 22 , 12–32. 10.1177/1066896913517939 (2014). Muiznieks, L. D. & Keeley, F. W. Molecular assembly and mechanical properties of the extracellular matrix: A fibrous protein perspective. Biochim. et Biophys. Acta (BBA)-Molecular Basis Disease . 1832 , 866–875 (2013). Kubanek, J. et al. Ultrasound modulates ion channel currents. Scientific Report s 6, 24170, doi: (2016). 10.1038/srep24170 Qiu, Z. et al. The Mechanosensitive Ion Channel Piezo1 Significantly Mediates In Vitro Ultrasonic of Neurons. iScience 21, 448–457, doi: (2019). 10.1016/j.isci.2019.10.037 Yoo, S., Mittelstein, D. R., Hurt, R., Lacroix, J. & Shapiro, M. G. Focused ultrasound excites neurons via mechanosensitive calcium accumulation and ion channel amplification. bioRxiv , 2020.2005.2019.101196, doi: (2020). 10.1101/2020.05.19.101196 Berrout, J., Jin, M. & O'Neil, R. G. Critical role of TRPP2 and TRPC1 channels in stretch-induced injury of blood-brain barrier endothelial cells. Brain Res. 1436 , 1–12. 10.1016/j.brainres.2011.11.044 (2012). Samanta, A., Hughes, T. E. T. & Moiseenkova-Bell, V. Y. Transient Receptor Potential (TRP) Channels. Subcell. Biochem. 87 , 141–165. 10.1007/978-981-10-7757-9_6 (2018). Li, H. T. R. P., Channel & Classification Adv. Exp. Med. Biol. 976 , 1–8, doi: 10.1007/978-94-024-1088-4_1 (2017). Christensen, A. P. & Corey, D. P. TRP channels in mechanosensation: direct or indirect activation? Nat. Rev. Neurosci. 8 , 510–521. 10.1038/nrn2149 (2007). Agarwala, S. S. & Kirkwood, J. M. Temozolomide, a novel alkylating agent with activity in the central nervous system, may improve the treatment of advanced metastatic melanoma. oncologist . 5 , 144–151. 10.1634/theoncologist.5-2-144 (2000). Ostermann, S. et al. Plasma and cerebrospinal fluid population pharmacokinetics of temozolomide in malignant glioma patients. Clin. cancer research: official J. Am. Association Cancer Res. 10 , 3728–3736. 10.1158/1078-0432.Ccr-03-0807 (2004). Berrocal, A. et al. Extended-schedule dose-dense temozolomide in refractory gliomas. Journal of neuro-oncology 96, 417–422, doi: (2010). 10.1007/s11060-009-9980-7 Doblas, S. et al. Glioma morphology and tumor-induced vascular alterations revealed in seven rodent glioma models by in vivo magnetic resonance imaging and angiography. J. Magn. Reson. Imaging . 32 , 267–275. 10.1002/jmri.22263 (2010). Doblas, S. et al. In vivo characterization of several rodent glioma models by 1H MRS. NMR Biomed. 25 , 685–694. 10.1002/nbm.1785 (2012). Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5202132","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":375906063,"identity":"0ace920b-1f2f-4548-9efb-97cc33c8a1be","order_by":0,"name":"Hyo Jin Choi","email":"","orcid":"","institution":"Daegu-Gyeongbuk Medical Innovation Foundation (K- MEDI hub)","correspondingAuthor":false,"prefix":"","firstName":"Hyo","middleName":"Jin","lastName":"Choi","suffix":""},{"id":375906064,"identity":"67cbf2bf-6311-4c8a-bfd2-43e604471319","order_by":1,"name":"Mun Han","email":"","orcid":"","institution":"Daegu-Gyeongbuk Medical Innovation Foundation (K- MEDI hub)","correspondingAuthor":false,"prefix":"","firstName":"Mun","middleName":"","lastName":"Han","suffix":""},{"id":375906065,"identity":"4acf68fb-2639-4a17-bdeb-d16c01d51d62","order_by":2,"name":"Byeongjin Jung","email":"","orcid":"","institution":"Daegu TechnoPark","correspondingAuthor":false,"prefix":"","firstName":"Byeongjin","middleName":"","lastName":"Jung","suffix":""},{"id":375906066,"identity":"c16d86e1-70dd-4408-90cb-21e762b01fec","order_by":3,"name":"Hyungkyu Huh","email":"","orcid":"","institution":"Daegu-Gyeongbuk Medical Innovation Foundation (K- MEDI hub)","correspondingAuthor":false,"prefix":"","firstName":"Hyungkyu","middleName":"","lastName":"Huh","suffix":""},{"id":375906067,"identity":"acdf0856-fa2e-46fa-81d9-db1a7511fc1c","order_by":4,"name":"Eun-hee Lee","email":"","orcid":"","institution":"Daegu-Gyeongbuk Medical Innovation Foundation (K- MEDI hub)","correspondingAuthor":false,"prefix":"","firstName":"Eun-hee","middleName":"","lastName":"Lee","suffix":""},{"id":375906068,"identity":"4db7ddb4-8210-4cb5-bdd0-f184b1929904","order_by":5,"name":"Jong-ryul Choi","email":"","orcid":"","institution":"Daegu-Gyeongbuk Medical Innovation Foundation (K- MEDI hub)","correspondingAuthor":false,"prefix":"","firstName":"Jong-ryul","middleName":"","lastName":"Choi","suffix":""},{"id":375906069,"identity":"3117e2b9-3e19-43d1-bd67-40e3417062bb","order_by":6,"name":"Juyoung Park","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5UlEQVRIiWNgGAWjYJACCRDBD8TMUAFm3GqRtUg2kKzF4ACxWvilmw/e+FBxz974/OKHnwsq7jDwtx9gNq7Ao0VyzrFkyxlnihO33XhmLD3jzDMGiTMJzIln8GgxuJFjJs3blpBgduOAGTNv22EGhhsMzAcb8GrJ/wbSYm884/g3sBZ5wlpy2EBaGDfw90BsMQBqScSnBegXY6BfEhJn3OApluY5c5jH8ExisyE+LcAQewgMsQR7/v7jGz/zVByWkzt++LAkPi2QSAEzEsAUDwMDI14NSFr4D+BXOApGwSgYBSMXAAC1dEu27RT17wAAAABJRU5ErkJggg==","orcid":"","institution":"Gachon University","correspondingAuthor":true,"prefix":"","firstName":"Juyoung","middleName":"","lastName":"Park","suffix":""}],"badges":[],"createdAt":"2024-10-04 07:23:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5202132/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5202132/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-88379-5","type":"published","date":"2025-02-24T15:57:50+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":70037281,"identity":"9edb0e98-a858-4c20-9bab-5b05b93aa17b","added_by":"auto","created_at":"2024-11-27 17:27:56","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":246539,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic of the MRgFUS system for BBBD. The sonication region was targeted using MRI and the transducer position was controlled in synchronization with the MRgFUS system.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-5202132/v1/7cec2a70bebb4484b61b948d.png"},{"id":70037283,"identity":"d59e6c0c-7cbd-485a-bfaa-cd9b66460bf5","added_by":"auto","created_at":"2024-11-27 17:27:56","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":462273,"visible":true,"origin":"","legend":"\u003cp\u003eExperimental timeline of \u003cstrong\u003e(A)\u003c/strong\u003e FUS protocol, \u003cstrong\u003e(B)\u003c/strong\u003e BBBD protocol and \u003cstrong\u003e(C)\u003c/strong\u003e FUS+BBBD protocol. Each bar indicates experimental steps from the anesthesia of the animal to the sacrifice. The blue and red box and text indicate FUS and BBBD sonication, respectively. (MB: Microbubbles, DOX: Doxorubicin, EB: Evans blue)\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-5202132/v1/4d516ccb0c43a5404265ccd8.png"},{"id":70037279,"identity":"51a030bd-b6ee-4e99-8114-56735b9bb9a8","added_by":"auto","created_at":"2024-11-27 17:27:56","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":574725,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Representative T1-weighted MR images for each protocol (“B”, “1.0FB”, and “1.0F”) taken before, and at 0, 3, 5, 7, 9, and 11 minutes after contrast agent injection. (\u003cstrong\u003eB)\u003c/strong\u003e A relative signal intensity changes at the sonicated region. Scale bars represent 5 mm. (Signal intensity: “B” n=4, Blue box, line, and circle; “1.0FB” n=4, Red box, line, and circle; “1.0F” n=4, Green box, line, and circle; “C” n=4, Yellow box, line, and circle). \u003cem\u003e*p \u003c/em\u003e\u0026lt; 0.05,\u003cem\u003e **p \u003c/em\u003e\u0026lt; 0.01,\u003cem\u003e ***p \u003c/em\u003e\u0026lt; 0.001,\u003cem\u003e ****p \u003c/em\u003e\u0026lt; 0.0001 vs. “C.” Values of \u003cem\u003ep \u0026lt; \u003c/em\u003e0.05 were considered as statistically significant.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-5202132/v1/49d97463a6b678af180c1010.png"},{"id":70038361,"identity":"9bf12c4e-c8ea-49dd-88b1-b29b4fa48f71","added_by":"auto","created_at":"2024-11-27 17:35:56","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":966178,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative K\u003csub\u003etrans\u003c/sub\u003e map of “B” (blue box), “1.0FB” (red box), and “1.0F” (green box) protocol and the contralateral region (yellow box) \u003cstrong\u003e(A)\u003c/strong\u003e before and \u003cstrong\u003e(B)\u003c/strong\u003e after sonication. The color bar indicates the BBB permeability. \u003cstrong\u003e(C)\u003c/strong\u003e The group averaged mean K\u003csub\u003etrans\u003c/sub\u003e value before and after the sonication. Scale bars represent 2 mm. (K\u003csub\u003etrans\u003c/sub\u003e value: “B” n=4, Blue box; “1.0FB” n=4, Red box; “1.0F” n=4, Green box; “C” n=4, Yellow box). \u003cem\u003e*p \u003c/em\u003e\u0026lt; 0.05,\u003cem\u003e **p \u003c/em\u003e\u0026lt; 0.01,\u003cem\u003e ***p \u003c/em\u003e\u0026lt; 0.001,\u003cem\u003e \u003c/em\u003evs. “C.” \u003cem\u003eP \u003c/em\u003e-values\u003cem\u003e\u0026lt; \u003c/em\u003e0.05 were considered statistically significant.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-5202132/v1/ca074032fd8c81af1c56fe96.png"},{"id":70038360,"identity":"61845a79-b582-4ae1-8b61-e650c1178f26","added_by":"auto","created_at":"2024-11-27 17:35:56","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":102557,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of the DOX quantification measured 24 hours after sonication using a fluorometric detector. The bars represent the average DOX concentration for each group (“C”, n=12; “B”, n=8; “0.5FB”, n=5; “1.0FB”, n=6, “2.0FB”, n=8; “1.0F”, n=5), while the dots represent individual cases. (\u003cem\u003e*p \u003c/em\u003e\u0026lt; 0.05)\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-5202132/v1/5b18cf74adc40b75e074bc92.png"},{"id":70037284,"identity":"e4cd4c07-ec51-4f1b-8c60-056532126240","added_by":"auto","created_at":"2024-11-27 17:27:56","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1345535,"visible":true,"origin":"","legend":"\u003cp\u003eHistopathological analysis of the rat brain tumor after the “B”, “1.0FB”, and “1.0F” with H\u0026amp;E staining. The images indicate representative photomicrographs of “B”, “1.0FB”, and “1.0F”. The 5× and 20× magnified images were obtained from a whole-brain section specimen. Scale bars represent 50 mm at 5× magnified images and 100 mm at 20× magnified images, respectively. (“B”, Blue box; “1.0FB”, Red box; “1.0F”, Green box)\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-5202132/v1/6815dc7cc516c5bc66df19ae.png"},{"id":77622532,"identity":"3804039c-339d-4a3f-97ec-5fe30d16117c","added_by":"auto","created_at":"2025-03-03 16:08:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5442744,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5202132/v1/f2531486-ae39-417d-902b-0af87c2ab3c9.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Augmented Blood-Tumor Barrier Permeability and Enhanced Doxorubicin Delivery in Rat Brain Tumor Models Through Additional Focused Ultrasound Stimulation","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe blood-brain barrier (BBB) is an obstacle to the effective treatment of brain tumors because it limits drug delivery and penetration\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Vigorous angiogenesis in tumors forms a heterogeneous vasculature with a compromised BBB, the so-called blood-tumor barrier (BTB)\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Due to its permeability, the BTB allows for the extravasation of small and large molecules \u003csup\u003e\u003cspan additionalcitationids=\"CR5 CR6\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. However, the BTB permeability is highly heterogeneous depending on the type, stage, and lesion of the brain tumor. Hence, the controlled delivery of adequate amounts of therapeutic agents for tumor treatment is still unclear\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eRadiation therapy combined with chemotherapy is currently used as the international standard of care for brain tumor treatment. Alternative methods, such as mannitol and bradykinin, have been attempted to increase the amount of therapeutic agent delivery for effective tumor treatment\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. However, limitations such as unpredictable BBB disruption can pose a potential risk to normal brain tissue\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Various experimental approaches, including the use of viral vectors\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e, polymeric nanoparticles\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, and exosomes\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e, have also been investigated to improve drug delivery across the BBB and BTB (hereafter referred to as BBB/BTB)\u003csup\u003e14\u003c/sup\u003e. Unfortunately, these methods are not yet used in clinical practice owing to some limitations such as difficulties in manufacturing, high production cost, safety\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e and therapeutic efficacy due to low accumulation\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFocused ultrasound (FUS) combined with intravenously injected microbubbles (MBs) has recently emerged as a promising non-invasive approach for disrupting the BBB/BTB. Preclinical studies using glioma models in animals have demonstrated its potential to enhance the delivery of therapeutic agents, including trastuzumab\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, doxorubicin\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, temozolomide\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, methotrexate\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e, and carboplatin\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, to targeted brain tumor regions. These studies have shown tumor suppression and improved survival rates in rat and mouse brain tumor models. Additionally, several clinical studies have confirmed the safety and feasibility of focused ultrasound BBB disruption (FUS-BBBD) using various devices, multiple treatment sessions, and various chemotherapies in patients with newly diagnosed GBM, recurrent GBM, and high-grade gliomas\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Despite the progress made in preclinical and clinical trials, further optimization of FUS parameters, including exposure settings and MB doses, is needed to minimize the risk of hemorrhage and other adverse effects while maximizing therapeutic outcomes\u003csup\u003e\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWe previously reported that an additional FUS stimulation (\u0026ldquo;FUS protocol\u0026rdquo;) step without MB injection prior to the conventional BBBD (\u0026ldquo;BBBD protocol\u0026rdquo;) enhances BBB permeability and doxorubicin (DOX) delivery to the brain of healthy rats without damaging nearby tissues or vessels \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. However, the robustness of this new FUS protocol (\u0026ldquo;FUS\u0026thinsp;+\u0026thinsp;BBBD protocol\u0026rdquo;) has not been validated in the tumor microenvironment. As a follow-up study, we tested the efficacy and efficiency of the FUS-BBBD protocol in the tumor rat model.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals and husbandry\u003c/h2\u003e \u003cp\u003e All experiments were approved by the Animal Experiment Ethics Committee of Daegu\u0026ndash;Gyeongbuk Medical Innovation Foundation (IACUC No. DGMIF-20122901-00). All procedures were conducted in accordance with the relevant guidelines. The study was also conducted in compliance with the ARRIVE (Animal Research: reporting of in vivo experiments) guidelines (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://arriveguidelines.org/arrive-guidelines\u003c/span\u003e\u003cspan address=\"https://arriveguidelines.org/arrive-guidelines\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). All efforts were made to minimize the number of animals and their pain.\u003c/p\u003e \u003cp\u003eMale Sprague-Dawley rats (5 weeks old, weighing 100\u0026ndash;150 g for tumor drug delivery study, n\u0026thinsp;=\u0026thinsp;35) were purchased from Orient Bio Inc. (Sungnam, Republic of Korea). All rats were used after 7 days of acclimatization in the vivarium of the Laboratory Animal Center (LAC) of the Daegu-Gyeongbuk Medical Innovation Foundation (DGMIF). Rats were fed a normal diet with water during acclimatization. The temperature (20\u0026ndash;25\u0026deg;C) and humidity (40\u0026ndash;45%) were controlled, and a 12:12 h light/dark cycle in polycarbonate cages. The rats used in this study were randomly divided into five groups based on the sonication protocol (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSummary of the sonication protocol groups.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eExperimental purpose\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSonication protocol\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNo of sonication targets\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"12\" rowspan=\"13\"\u003e \u003cp\u003e\u003cb\u003eTumor model\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eBBB permeablization\u003c/p\u003e \u003cp\u003e(T1 and DCE-MRI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCtrl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.0F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.0FB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"5\" rowspan=\"6\"\u003e \u003cp\u003eDrug delivery\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCtrl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.0F\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eB\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.5FB\u003csup\u003e***\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.0FB\u003csup\u003e****\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.0FB\u003csup\u003e*****\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eHistopathology\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.0F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eB\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.0FB\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cb\u003eNormal\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eImmunostaining\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.0F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eB\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.0FB\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003e\u003csup\u003e*\u003c/sup\u003e1.0F: 1.0MPa w/o MB; \u003csup\u003e**\u003c/sup\u003eB: 0.72MPa w/ MB; \u003csup\u003e***\u003c/sup\u003e0.5FB: 0.5MPa w/o MB\u0026thinsp;+\u0026thinsp;0.72MPa w/ MB; \u003csup\u003e****\u003c/sup\u003e1.0FB: 1.0MPa w/o MB\u0026thinsp;+\u0026thinsp;0.72MPa w/ MB; \u003csup\u003e*****\u003c/sup\u003e2.0FB: 2.0MPa w/o MB\u0026thinsp;+\u0026thinsp;0.72MPa w/ MB\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eReagents\u003c/h3\u003e\n\u003cp\u003eDoxorubicin hydrochloride (DOX) (Boryung Pharmaceutical, Seoul, Republic of Korea) was used to confirm drug delivery in the tumor environment. Evans blue (Sigma-Aldrich St. Louis, MO, USA) was used to demonstrate the BBB opening region. Zoletil (Virbac Laboratories, Carros, France) and Rompun (Bayer, Leverkusen, Germany) were used for animal anesthesia. Microbubbles (MBs) Definity\u0026reg; were purchased (Lantheus Medical Imaging, North Billerica, MA, USA) for BBB opening along with FUS, and gadoterate meglumine (Gd-DOTA) was purchased (Dotarem\u003csup\u003e\u0026reg;\u003c/sup\u003e; Guerbet, Roissy CDG, France) to identify the BBB opening region using MRI.\u003c/p\u003e\n\u003ch3\u003e9L gliosarcoma cell culture\u003c/h3\u003e\n\u003cp\u003e9L gliosarcoma cell lines (ATCC\u0026reg; CRL-2200\u003csup\u003e\u0026trade;\u003c/sup\u003e) were cultured at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e in Dulbecco Modified Eagle Medium (DMEM; 11995-073, Gibco, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (FBS; 16000-044, Gibco, Gaithersburg, MD, USA) and 1% penicillin-streptomycin (PS; 15140-122, Gibco, Gaithersburg, MD, USA), as previously described\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. At approximately 90% cell confluence, the media was removed and the cells were removed using 0.25% trypsin-EDTA (25200-056, Gibco, Gaithersburg, MD, USA). To prepare the tumor cells for implantation, they were centrifuged at 1,000 rpm for 2 min, suspended in DMEM at a final concentration of 5\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/5 \u0026micro;L, and kept cool until implantation. Before tumor implantation, the 9L gliosarcoma stock was thawed and used for the experiment after the acclimatization period. All brain tumor implantations were carried out with 9L gliosarcoma at passage number 5\u0026ndash;7.\u003c/p\u003e\n\u003ch3\u003eBrain tumor implantation procedure\u003c/h3\u003e\n\u003cp\u003eThe rats were anesthetized with an intramuscular injected anesthetic mixture [Zoletil (25 mg/kg): Rompun (4.6 mg/kg): saline\u0026thinsp;=\u0026thinsp;5:2:3] prior to tumor implantation. The rat was fixed to the stereotaxic frame (Harvard Apparatus, Holliston, MA, USA) using an in-ear and upper tooth bar for hemostasis. Animal hair was shaved, and a sagittal incision was made on the posterior aspect of the head, approximately 2 cm. A small burr hole was drilled using a dental drill (Saeshin, Daegu, Korea) 3 mm lateral and 1 mm anterior to the \u003cem\u003ebregma\u003c/em\u003e. The 9L gliosarcoma cells filled in a 25 \u0026micro;l Hamilton syringe (Hamilton Company, Reno, NV, USA) were slowly injected at 5 mm under the dura mater at a flow rate of 1 \u0026micro;L/min for 5 min using a 30 gauge Hamilton needle (Hamilton Company, Reno, NV, USA) and a syringe pump (Harvard Apparatus, Holliston, MA, USA). To avoid any cell leakage or backflow of the media, holes were filled with Hy-bond polycarboxylate cement (Shofu, TYOKO, Japan) and the skin was sutured. After 10\u0026ndash;14 days of implantation, tumor size was checked using a horizontal bore 9.4T animal MRI (BioSpec 94/20 USR; Bruker, Billerica, MA, USA) for controlled experiments. Total 35 rats that presented with a tumor diameter of 1.5\u0026thinsp;~\u0026thinsp;2.0 mm based on T2-weighted (T2W) coronal images were selected and used in the experiment.\u003c/p\u003e\n\u003ch3\u003eMRI-guided Focused Ultrasound (MRgFUS) system\u003c/h3\u003e\n\u003cp\u003eThe MRgFUS system (RK-100; FUS Instruments, Toronto, Canada) was used to sonicate rat brains for the BBB as described previously \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. A schematic of the system is presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Briefly, the system has an air-back, single-element, and spherical piezoelectric transducer (FUS Instruments, Toronto, Canada). A spherical piezoelectric transducer with a diameter of 75 mm and a radius of curvature of 60 mm, which generates a resonant frequency of 1 MHz, was used. The ultrasound \u0026minus;\u0026thinsp;3 dB acoustic pressure width and length of the focal region were 1.5 mm and 6 mm in free water at the peak pressure measured by the Acoustic Intensity Measurement System (AIMS III, ONDA, Sunnyvale, CA, USA) and the hydrophone (HGL-400, ONDA, Sunnyvale, CA, USA)\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. The transducer was driven by a waveform generator (33220A; Agilent, Santa Clara, CA, USA) and an RF power amplifier (4010L; E\u0026amp;I, Rochester, NY, USA). The transducer was submerged in a degassed water tank. A 9.4T MRI and a volume coil with an 86-mm inner diameter were used for image guidance by syncing the coordinates between the two systems (MRI and MRgFUS system).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eSonication protocol\u003c/h2\u003e \u003cp\u003eSonication was performed using the MRgFUS system, as described in section 2.5. Burst sonication (10ms tone burst, pulse repetition frequency (PRF) of 1Hz for 120 s) was performed at the location of the tumor. The acoustic pressure of the FUS protocol \u0026ldquo;F\u0026rdquo; was 0.5, 1.0, and 2.0 MPa depending on the experimental purpose (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) and the acoustic pressure of the BBBD protocol \u0026ldquo;B\u0026rdquo; was 0.72 MPa. The FUS\u0026thinsp;+\u0026thinsp;BBBD protocol \u0026ldquo;FB\u0026rdquo; was a fusion of both, where \u0026ldquo;FUS protocol\u0026rdquo; ultrasound was applied prior to the \u0026ldquo;BBBD protocol\u0026rdquo; without MB injection. The details of the experimental schedule for the sonication protocols are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. These sonication parameters have been verified as safe conditions in previous studies\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Although BBB/BTB is simultaneously disrupted in the tumor animal model, protocol used in this study is referred as BBBD throughout the manuscript in order to minimize confusion and to be consistent with previous studies.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMagnetic resonance imaging\u003c/h3\u003e\n\u003cp\u003eA 9.4T preclinical animal MRI was used for magnetic resonance imaging. T1-weighted (T1W) MR images were used to check the BBB disruption. T2-weighted (T2W) MR images were used as a guide for ultrasound targeting and tumor size checking. Meanwhile, DCE-MRI was used to identify the permeability\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. MRI parameters were as follows: T1W, echo time (TE)\u0026thinsp;=\u0026thinsp;6.5 ms, repetition time (TR)\u0026thinsp;=\u0026thinsp;1500 ms, field of view (FOV) of 40 \u0026times; 40 mm\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, matrix size of 256 \u0026times; 256, axial and coronal slices of 1.5 mm slice thickness without a gap, and the number of excitations (NEX)\u0026thinsp;=\u0026thinsp;3. T2W: TE\u0026thinsp;=\u0026thinsp;33 ms, TR\u0026thinsp;=\u0026thinsp;2500 ms, FOV\u0026thinsp;=\u0026thinsp;40 \u0026times; 40 mm\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, matrix size\u0026thinsp;=\u0026thinsp;256 \u0026times; 256, axial and coronal slices of 1.5 mm slice thickness without a gap, and NEX\u0026thinsp;=\u0026thinsp;2. DCE-MRI: TE\u0026thinsp;=\u0026thinsp;1.5 ms, TR\u0026thinsp;=\u0026thinsp;24.3 ms, FOV\u0026thinsp;=\u0026thinsp;40 \u0026times; 40 mm\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, matrix size of 128 \u0026times; 128, axial and coronal slices of 1.5 mm slice thickness without a gap, and NEX\u0026thinsp;=\u0026thinsp;3. The MRI parameters are summarized in Table. 2.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMR Imaging Parameters.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026times;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026times;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSequence\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUse\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTE\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e(ms)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTR\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e(ms)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eFOV\u003csup\u003e***\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e(mm\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMatrix\u003c/p\u003e \u003cp\u003eSize\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eSlice\u003c/p\u003e \u003cp\u003eThickness\u003c/p\u003e \u003cp\u003e(mm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNEX\u003csup\u003e****\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRARE\u003c/p\u003e \u003cp\u003eT1-weighted\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDetection of BBB disruption\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026times;\" colname=\"c5\"\u003e \u003cp\u003e40\u0026times;40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026times;\" colname=\"c6\"\u003e \u003cp\u003e256\u0026times;256\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRARE\u003c/p\u003e \u003cp\u003eT2-weighted\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSonication target planning/ Detection of edema/\u003c/p\u003e \u003cp\u003eTumor size checking\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026times;\" colname=\"c5\"\u003e \u003cp\u003e40\u0026times;40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026times;\" colname=\"c6\"\u003e \u003cp\u003e256\u0026times;256\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFLASH DCE-MRI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePermeability measurement\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e24.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026times;\" colname=\"c5\"\u003e \u003cp\u003e40\u0026times;40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026times;\" colname=\"c6\"\u003e \u003cp\u003e128\u0026times;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"8\"\u003e\u003csup\u003e*\u003c/sup\u003eTE: Echo time; \u003csup\u003e**\u003c/sup\u003eTR: Repetition time; \u003csup\u003e***\u003c/sup\u003eFOV: Field of view; \u003csup\u003e****\u003c/sup\u003eNEX: Number of excitations\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"8\"\u003eThe region of interest (ROI), which is the focal area of the transducer, was selected as a circle with a diameter of 1.5 mm. The relative signal intensity \u003cem\u003eR\u003c/em\u003e\u003csub\u003e\u003cem\u003et\u003c/em\u003e\u003c/sub\u003e of the tumor region at a given time point (T1\u003csub\u003et\u003c/sub\u003e) was normalized by the signal intensity of pre-injection T1W MR images (0 min, T1\u003csub\u003epre\u003c/sub\u003e) using the following equation:\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003cdiv id=\"Equa\" class=\"Equation\"\u003e \u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:{R}_{t}\\left(\\text{\\%}\\right)=\\:\\frac{\\left({T1}_{t}-{T1}_{pre}\\right)}{{T1}_{pre}}\\text{*}100$$\u003c/div\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe BBB permeability K\u003csub\u003etrans\u003c/sub\u003e was calculated based on a Patlak model using DCE-MRI images, similar to a previous study\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. T1 measurements were acquired using (RARE VTR) (TR\u0026thinsp;=\u0026thinsp;160, 180, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1500, 1800, 2000, 2500, 3000, 3500, 4000, 6000, and 12000 ms). Ten sets of FLASH images were acquired before the contrast agent injection (Gd-DOTA with relaxivity of 3.0 s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003emM\u003csup\u003e\u0026minus;\u003c/sup\u003e), followed by 90 sets of FLASH images acquired during and after CA injection with a temporal resolution of 9.32 s for 10 min. The same slice position was used for T1 mapping and DCE-MRI. The ROI was selected using a 5-pixel circle diameter, which corresponds to a 1.5 mm focal area of the transducer (3.2 pixels/mm). The plasma concentration of the contrast agent was measured in a large blood vessel (ophthalmic artery or transverse sinus). The hematocrit level was assumed to be 45% in the capillary\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003ch3\u003eBlood-brain barrier disruption experiment\u003c/h3\u003e\n\u003cp\u003eThe blood-brain barrier and blood-tumor barrier disruption experiments were performed as described in our previous studies\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Before sonication, the tumor size was checked with T1-weighted MR images 10\u0026ndash;14 days after tumor implantation. A tumor diameter of 1.5\u0026thinsp;~\u0026thinsp;2.0 mm was selected and used in this study. Briefly, rats were anesthetized with an intramuscular injection of an anesthetic mixture (Zoletil (25 mg/kg):Rompun (4.6 mg/kg): saline\u0026thinsp;=\u0026thinsp;5:2:3). After anesthesia, an angiocatheter was inserted into the vein of the tail, and the hair on the head was shaved. Then, the rat was placed in a supine position on an MR-compatible plastic bed, and the exposed scalp was immersed in a degassed water bag using fixed ear and tooth bars (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Sonication of the tumor region was performed according to the protocol described in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The T2W MR image was scanned and registered to the MRgFUS system and placed in sync. The 0.02 mL/kg perflutren lipid MB (Definity\u0026reg;) were diluted 1:50 in saline and administered through an angiocatheter using an automated syringe pump (Harvard Apparatus, Holliston, MA, USA). Sonication was then started 10 s after the diluted MB solution infusion. Following sonication, T1W MR or DCE-MR images were performed to identify BBBD and permeability with Gd-DOTA (0.2 mM/kg). DOX (5.67 mg/kg) and Evans blue (4 mg/kg) were administered intravenously (i.v.) to mark the DOX delivered region for tissue extraction.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eDoxorubicin quantification\u003c/h2\u003e \u003cp\u003eAll rats were sacrificed 24 h after the IV administration of DOX and perfused with 0.9% NaCl via transcardial perfusion followed by brain extraction. Then, the sample volume (approximately 30 mg of tissue) of the tumor region was harvested for quantification. The tumor samples were homogenized with acidified ethanol (50% ethanol in 0.3 N HCl) using a bead beater and incubated for 24 h at 4\u0026deg;C. After incubation, the samples were centrifuged at 16,000 \u0026times; g for 20 min to remove the tissue pellet, and the supernatant was collected. The fluorescence intensity was measured using a fluorometric detector with 480 nm excitation and 590 nm emission (Infinite 200 Pro; TECAN, Austria GmbH, Austria). The concentration of DOX was quantified using a standard curve derived from eight serial concentrations of pure DOX (0, 10, 20, 50, 100, 200, 500, and 1000 ng/mL). Fluorescence intensity was analyzed using TECAN i-control software (TECAN, Austria GmbH, Austria), and all measurements were repeated at least three times. The contralateral tumor region without sonication was used as a control to correct autofluorescence. DOX fluorescence detection and analysis were performed as described previously\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eHematoxylin and Eosin (H\u0026amp;E) staining\u003c/h2\u003e \u003cp\u003eHistopathological changes in the sonicated rat brain tissue were investigated by H\u0026amp;E staining as described previously\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. The sonicated rats were sacrificed 24 hours post-sonication, followed by transcardiac perfusion with 0.9% NaCl and fixation with 10% formalin. The rat brains were extracted and fixed in 10% formalin for 7 days. The brain tissue samples were then embedded in paraffin and cut serially in 4 \u0026micro;m thickness axial direction using a microtome (HistoCore AUTOCUT; Leica, Wetzlar, Germany). Paraffin-embedded sections were deparaffinized and rehydrated before staining. Brain tissue slides were stained with an H\u0026amp;E stain kit (Vector Laboratories, Burlingame, CA, USA). Images were taken with a digital slide scanner (Axio Scan.Z1; Carl Zeiss, Oberkochen, Germany).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eThe values obtained were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Statistical significance differences between the control group and experimental groups were compared using a two-tailed paired t-test, one-way ANOVA with Tukey\u0026rsquo;s test, and two-way ANOVA with Tukey\u0026rsquo;s test using GraphPad Prism 8 (GraphPad Software, Inc., San Diego, CA, USA). Statistical significance was accepted for \u003cem\u003ep\u003c/em\u003e-values\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eBBB/BTB permeability\u003c/h2\u003e \u003cp\u003eTo investigate the effect of the additional FUS stimulation on the BBB/BTB permeability, contrast-enhanced T1W MR imaging was performed for \u0026ldquo;B\u0026rdquo;, \u0026ldquo;1.0FB\u0026rdquo;, and \u0026ldquo;1.0F\u0026rdquo; as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Representative MR Images of each case show the average signal intensity of the contrast agent after BBB/BTB disruption (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). For every given case, averaged signal intensity of the sonicated tumor region was higher than the non-sonicated contralateral \u0026ldquo;C\u0026rdquo; region in both \u0026ldquo;B\u0026rdquo; and \u0026ldquo;1.0FB\u0026rdquo; cases. However, this difference was not observed for the \u0026ldquo;1.0F\u0026rdquo; case. To quantify the BBB/BTB permeability, the relative signal intensity of the contrast agent was measured in a time-dependent manner (before, 0, 3, 5, 7, 9, and 11 min), as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB. The relative signal intensity of the target area increased by 33.2\u0026thinsp;\u0026plusmn;\u0026thinsp;3.1%, 41.2\u0026thinsp;\u0026plusmn;\u0026thinsp;3.1%, 43.9\u0026thinsp;\u0026plusmn;\u0026thinsp;3.3%, 45.9\u0026thinsp;\u0026plusmn;\u0026thinsp;3.0%, 44.9\u0026thinsp;\u0026plusmn;\u0026thinsp;2.7% and 55.0\u0026thinsp;\u0026plusmn;\u0026thinsp;4.9%, 54.5\u0026thinsp;\u0026plusmn;\u0026thinsp;3.4%, 51.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3%, 48.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7%, 47.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1% for each time point compared to the contralateral region under \u0026ldquo;B\u0026rdquo; and \u0026ldquo;1.0FB\u0026rdquo; protocol, respectively. However, the \u0026ldquo;1.0F\u0026rdquo; region showed no significant change compared to the contralateral \u0026ldquo;C\u0026rdquo; region. In these results, the relative signal intensity was highest (2.65 times compared to the contralateral region) at the \u0026ldquo;1.0FB\u0026rdquo; region, 3 min after the MR contrast agent injection.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn addition to the averaged signal intensity changes, brain permeability (K\u003csub\u003etrans\u003c/sub\u003e) was calculated using DCE-MRI for each protocol (\u0026ldquo;B\u0026rdquo;, \u0026ldquo;1.0FB\u0026rdquo;, and \u0026ldquo;1.0F\u0026rdquo;). The representative K\u003csub\u003etrans\u003c/sub\u003e map and group-averaged K\u003csub\u003etrans\u003c/sub\u003e values before and after the sonication procedure are shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB. The average permeability of the tumor region (n\u0026thinsp;=\u0026thinsp;4) sonicated by \u0026ldquo;B\u0026rdquo; and \u0026ldquo;1.0FB\u0026rdquo; protocol was enhanced by 0.0741\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0159 min\u003csup\u003e-1\u003c/sup\u003e and 0.0928\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0186 min\u003csup\u003e-\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e, respectively, which was 2.08-fold and 1.73-fold higher than that before sonication (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). The increase in K\u003csub\u003etrans\u003c/sub\u003e after sonication was statistically significant (p\u0026thinsp;=\u0026thinsp;0.031 and 0.0006 for \u0026ldquo;B\u0026rdquo; and \u0026ldquo;1.0FB\u0026rdquo; protocols, respectively. For the \u0026ldquo;1.0F\u0026rdquo; protocol, however, no significant increase in the brain permeability was observed (0.0379\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00701 min\u003csup\u003e-\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e after sonication, compared to 0.0378\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00738 min\u003csup\u003e-\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e before sonication with p\u0026thinsp;=\u0026thinsp;0.99).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eDOX delivery\u003c/h2\u003e \u003cp\u003eThe targeted brain tissue samples were measured for statistical comparison for DOX delivery for each protocol \u0026ldquo;B\u0026rdquo;, \u0026ldquo;1.0FB\u0026rdquo;, and \u0026ldquo;1.0F\u0026rdquo; as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. The average DOX concentration was 694.1\u0026thinsp;\u0026plusmn;\u0026thinsp;398.5 ng/g in the contralateral \u0026ldquo;C\u0026rdquo; region, while it was 885.1\u0026thinsp;\u0026plusmn;\u0026thinsp;305.6 ng/g in the tumor region sonicated with \u0026ldquo;B\u0026rdquo;, 996.2\u0026thinsp;\u0026plusmn;\u0026thinsp;408.0 ng/g for \u0026ldquo;0.5FB\u0026rdquo;, 1255.4\u0026thinsp;\u0026plusmn;\u0026thinsp;347.8 ng/g for \u0026ldquo;1.0FB\u0026rdquo;, 1327.8\u0026thinsp;\u0026plusmn;\u0026thinsp;470.7 ng/g for \u0026ldquo;2.0FB\u0026rdquo;, and 757.3\u0026thinsp;\u0026plusmn;\u0026thinsp;447.4 ng/g for \u0026ldquo;1.0F\u0026rdquo;. The amount of DOX delivered under the \u0026ldquo;B\u0026rdquo;, \u0026ldquo;0.5FB\u0026rdquo;, \u0026ldquo;1.0FB\u0026rdquo;, \u0026ldquo;2.0FB\u0026rdquo;, and \u0026ldquo;1.0F\u0026rdquo; protocols was 1.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44, 1.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.59, 1.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50, 1.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.68, and 1.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.64 times higher, respectively, than in the contralateral region without sonication \u0026ldquo;C\u0026rdquo;.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eHistopathological analysis\u003c/h2\u003e \u003cp\u003eH\u0026amp;E histopathological analysis (n\u0026thinsp;=\u0026thinsp;3) was performed to identify the damage caused by \u0026ldquo;B\u0026rdquo;, \u0026ldquo;1.0FB\u0026rdquo;, and \u0026ldquo;1.0F\u0026rdquo; in the brain tumor region. No significant differences between regions sonicated with each protocol were observed in the 5-and 20 times magnified images, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThere are increasing clinical demands\u003csup\u003e\u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e on safe and efficient drug delivery techniques for tumor treatment. A temporal BBB/BTB opening using focused ultrasound was suggested as a possible candidate\u003csup\u003e\u003cspan additionalcitationids=\"CR39\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Previous research demonstrated that incorporating an additional sonication step prior to conventional BBBD can enhance drug delivery in healthy rat brains by up to 1.75 times, without inducing tissue damage or heating\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. This additional sonication step, performed without microbubble injection, has shown to be a simple yet effective means of increasing drug delivery. The present study focuses on validating this protocol in tumor models, aiming to support its potential translation into clinical applications.\u003c/p\u003e \u003cp\u003eConsistent with previous experiments in normal rats, the addition of ultrasound sonication significantly enhanced BBB/BTB opening in the tumor environment. Given that the BTB is known to be more permeable than the intact blood-brain barrier\u003csup\u003e\u003cspan additionalcitationids=\"CR5 CR6\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e, baseline BTB permeability was assessed through MR imaging 24 hours prior to sonication to minimize experimental bias. Using the \u0026ldquo;1.0FB\u0026rdquo; protocol, the signal intensity of T1-weighted MR and K\u003csub\u003etrans\u003c/sub\u003e increased by approximately 2.42-fold and 2.08-fold, respectively, compared to pre-sonication measurements at the same target, and by 2.65-fold and 2.15-fold relative to the contralateral tumor region.\u003c/p\u003e \u003cp\u003eCompared to the conventional BBBD protocol \u0026ldquo;B\u0026rdquo;, the addition of ultrasound stimulation resulted in a 1.45-fold increase in BTB permeability. This is consistent with previous findings, where additional sonication prior to BBBD increased the average Ktrans in normal tissue by 1.36-fold compared to the conventional BBBD protocol\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Despite differences in the permeability characteristics of the BBB/BTB between the current tumor model and normal brain tissue, the addition of an extra sonication step prior to BBBD consistently produced a comparable increase in permeability.\u003c/p\u003e \u003cp\u003eThe amount of chemotherapeutic agent delivered was also compared after sonication using each protocol (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). In order to minimize experimental bias, animals with a tumor size of less than 20% difference were selected and used (and tumor diameter between 1.5\u0026thinsp;~\u0026thinsp;2.0 mm). The delivery of DOX to the target tumor region sonicated using the \u0026ldquo;1.0FB\u0026rdquo; protocol was 1.44-fold higher than that achieved with the conventional BBBD protocol \u0026ldquo;B\u0026rdquo;. The observed patterns in permeability and drug delivery were similar to those reported in a previous study\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, although the differences were not statistically significant. It is important to note that the previous study involved opening an intact BBB in a normal animal model, while the BTB in the brain tumor model used in this study was already more permeable and heterogeneous\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Additionally, the untreated contralateral brain tumor region exhibited variability in integrity due to existing BBB dysfunction. Both contrast agents and DOX showed heterogeneous permeability, even in the absence of ultrasound, which likely influenced the statistical significance of the findings\u003csup\u003e\u003cspan additionalcitationids=\"CR44\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAn additional sonication step prior to BBBD was shown to enhance BBB/BTB permeability, thereby increasing the overall delivery of chemotherapeutic agents in the tumor model. To assess the safety of this new protocol in the brain tumor environment, histopathological examination of brain tissue was performed using standard H\u0026amp;E staining to evaluate potential brain damage\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e,\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e no significant differences were observed in the \u0026ldquo;1.0FB\u0026rdquo; group compared to the \u0026ldquo;B\u0026rdquo; and \u0026ldquo;1.0F\u0026rdquo; groups.\u003c/p\u003e \u003cp\u003eThe enhancement of brain permeability by adding an extra sonication step prior to conventional BBBD (ultrasound with intravenously injected microbubbles) may increase sensitivity to acoustic energy by affecting the cytoskeleton and extracellular matrix through dynamic remodeling and matrix degradation\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. Another possibility is that the increased permeability results from the expansion of extracellular and perivascular spaces, rather than changes in the BBB itself. Recent studies have highlighted the role of mechanical stress on mechanosensitive calcium ion channels\u003csup\u003e\u003cspan additionalcitationids=\"CR50 CR51\" citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. Among these, TRP channels are widely expressed and activated by various stimuli, with the mammalian TRP channel family divided into six subfamilies: canonical (TRPC1-7), vanilloid (TRPV1-6), melastatin (TRPM1-8), polycystin (TRPP1-5), mucolipin (TRPML1-3), and ankyrin (TRPA1)\u003csup\u003e\u003cspan additionalcitationids=\"CR54\" citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. These ion channels are known to compromise BBB integrity, suggesting that prior FUS stimulation may enhance BBB disruption, potentially explaining the current findings\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. Further studies are required to fully understand the underlying mechanism of the drug delivery enhancement.\u003c/p\u003e \u003cp\u003eDOX is widely used in chemotherapeutic drug delivery studies. However, other therapeutic agents that could benefit from enhanced drug delivery were not evaluated in this study. For instance, TMZ is the primary treatment option for gliomas. It is a relatively small lipophilic molecule that can cross the BBB\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. It has been shown that TMZ levels in the brain and cerebrospinal fluid reach up to 20% of the drug plasma concentration\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. However, the therapeutic potential of TMZ for glioma treatment is limited by its short half-life (1.8 h). This requires continuous drug administration to maintain the therapeutic concentration of the drug in tumor tissue and to optimize therapeutic potential\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. The proposed FUS\u0026thinsp;+\u0026thinsp;BBBD protocol may offer a safe and effective method for delivering TMZ in the near future. In addition, only a well-characterized 9L gliosarcoma that is both highly immunogenic and poorly infiltrative was used in this study\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e,\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. Future studies should include a broader range of brain tumor models, such as glioma, glioblastoma, medulloblastoma, and brain metastasis, as each tumor type may present different characteristics due to tumor heterogeneity\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Finally, further study is required to validate the effect of mechanosensitive on calcium ion channels\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e,\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eConcluding remarks\u003c/h2\u003e \u003cp\u003eIn conclusion, the additional mechanical stress from ultrasound stimulation prior to conventional BBBD improves overall permeability in the target tumor region, leading to enhanced drug delivery. The proposed advanced \u0026ldquo;FUS\u0026thinsp;+\u0026thinsp;BBBD protocol\u0026rdquo; represents a promising strategy to increase the delivery of chemotherapeutic agents with minimal side effects in brain tumor treatment. Further research is necessary to elucidate the biomolecular and cellular mechanisms underlying the improved BBBD achieved with this protocol, both in preclinical studies and potential clinical applications.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by the Korea Health Technology R\u0026amp;D Project through the Korea Health Industry Development Institute, funded by the Ministry of Health \u0026amp; Welfare, Republic of Korea (Grant number: RS-2023-KH135060, and RS-2024-00337463). Also, this research was support by the Starting growth Technological R\u0026amp;D Program, funded by the Ministry of SMEs and Startups, Republic of Korea (Grant number: RS-2023-00261874). In addition, we would like to appreciate Yuri Hong who supported biological assays in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHyo Jin Choi:\u003c/strong\u003e Methodology, Histopathological sample preparation, Analysis and validation, Writing\u0026mdash;draft preparation, Writing\u0026mdash;review and editing\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMun Han:\u003c/strong\u003e Methodology, \u003cem\u003eIn vivo\u003c/em\u003e preclinical experiment, Analysis and validation, Writing\u0026mdash;draft preparation, Writing\u0026mdash;review and editing\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eByeongjin Jung:\u003c/strong\u003e Methodology, \u003cem\u003eIn vivo\u003c/em\u003e preclinical experiment, Writing\u0026mdash;draft preparation\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHyungkyu Huh:\u003c/strong\u003e Analysis and validation, Writing\u0026mdash;draft preparation\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEun-hee Lee:\u003c/strong\u003e Analysis and validation, Writing\u0026mdash;review and editing\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eJong-ryul Choi:\u003c/strong\u003e Analysis and validation, Writing\u0026mdash;review and editing\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eJuyoung Park:\u003c/strong\u003e Supervision, Conceptualization, Funding acquisition, Methodology, Analysis and validation, Writing\u0026mdash;draft preparation, Writing\u0026mdash;review and editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article. The datasets used and/or analyzed during the current study are also available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eDeeken, J. F. \u0026amp; Loscher, W. The blood-brain barrier and cancer: transporters, treatment, and Trojan horses. \u003cem\u003eClin. cancer research: official J. Am. Association Cancer Res.\u003c/em\u003e \u003cb\u003e13\u003c/b\u003e, 1663\u0026ndash;1674. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1158/1078-0432.ccr-06-2854\u003c/span\u003e\u003cspan address=\"10.1158/1078-0432.ccr-06-2854\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2007).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAchrol, A. S. et al. 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Despite its potential, optimizing FUS to maximize drug delivery while minimizing adverse effects remains a significant challenge. In this study, we evaluated a novel FUS protocol that incorporates additional FUS stimulation without microbubbles (MBs) (\"FUS protocol\") prior to conventional BBB disruption with MBs (\"BBBD protocol\") in a rat brain tumor model (n\u0026thinsp;=\u0026thinsp;35). This approach aimed to validate its effectiveness in enhancing BBB/BTB disruption and facilitating doxorubicin delivery. T1-weighted contrast-enhanced and dynamic contrast-enhanced (DCE) MRI demonstrated significant increases in signal intensity and permeability (K\u003csub\u003etrans\u003c/sub\u003e) in the tumor region under the \"FUS\u0026thinsp;+\u0026thinsp;BBBD protocol\u0026rdquo;, with 2.65-fold and 2.08-fold increases, respectively, compared to the non-sonicated contralateral region. These values were also elevated compared to the conventional \"BBBD protocol\" by 1.45-fold and 1.25-fold, respectively. Furthermore, doxorubicin delivery in the targeted region increased by 1.91-fold under the \"FUS\u0026thinsp;+\u0026thinsp;BBBD protocol\u0026rdquo;, compared to a 1.44-fold increase using the conventional \"BBBD protocol\u0026rdquo;. This novel FUS approach offers a promising, cost-effective strategy for enhancing drug delivery to brain tumors. While further studies are required to assess its applicability with different chemotherapeutics and tumor types, it holds significant potential for improving brain tumor treatment in both preclinical and clinical settings.\u003c/p\u003e","manuscriptTitle":"Augmented Blood-Tumor Barrier Permeability and Enhanced Doxorubicin Delivery in Rat Brain Tumor Models Through Additional Focused Ultrasound Stimulation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-27 17:27:51","doi":"10.21203/rs.3.rs-5202132/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-11-25T10:26:40+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-21T14:46:06+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-18T18:55:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"296817736394545195972661792949075188205","date":"2024-11-12T18:09:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"45098606686373886283715873266404397696","date":"2024-11-12T08:40:33+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-11-06T08:42:48+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-11-06T08:37:35+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-11-04T13:20:53+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-10-30T16:20:47+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-10-04T07:07:44+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"bf934603-1022-4967-843b-be9feb048384","owner":[],"postedDate":"November 27th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":40024360,"name":"Biological sciences/Cancer"},{"id":40024361,"name":"Biological sciences/Neuroscience"},{"id":40024362,"name":"Physical sciences/Energy science and technology"}],"tags":[],"updatedAt":"2025-03-03T16:02:15+00:00","versionOfRecord":{"articleIdentity":"rs-5202132","link":"https://doi.org/10.1038/s41598-025-88379-5","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-02-24 15:57:50","publishedOnDateReadable":"February 24th, 2025"},"versionCreatedAt":"2024-11-27 17:27:51","video":"","vorDoi":"10.1038/s41598-025-88379-5","vorDoiUrl":"https://doi.org/10.1038/s41598-025-88379-5","workflowStages":[]},"version":"v1","identity":"rs-5202132","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5202132","identity":"rs-5202132","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}