Brain Delivery of Cisplatin Using Microbubbles in Combination with Ultrasound as an Effective Therapy for Glioblastoma.

Glioblastoma is a highly invasive and fatal disease. Temozolomide, a blood-brain barrier (BBB)-penetrant therapeutic agent currently used for glioblastoma, does not exhibit sufficient therapeutic effect. Cisplatin (CDDP), a versatile anticancer drug, is not considered a therapeutic option for glioblastoma due to its low BBB permeability. We previously investigated the utility of microbubbles (MBs) in combination with ultrasound (US) in promoting BBB permeability and reported the efficacy of drug delivery to the brain using a minimally invasive approach. This study aimed to evaluate the feasibility of CDDP delivery to the brain using the combination of MBs and US for the treatment of glioblastoma. We used mice that were implanted with glioma-261 GFP-Luc cells expressing luciferase as the glioblastoma model. In this model, after tumor inoculation, the BBB opening was induced using MBs and US, and CDDP was simultaneously administered. We found that the CDDP concentrations were higher at the glioblastoma site where the US was applied, although CDDP normally cannot pass through the BBB. Furthermore, the survival was longer in mice treated with CDDP delivered via MBs and US than in those treated with CDDP alone or those that were left untreated. These results suggest that the combination of MBs and US is an effective antitumor drug delivery system based on BBB opening in glioblastoma therapy.

Development of a Gene and Nucleic Acid Delivery System for Skeletal Muscle Administration via Limb Perfusion Using Nanobubbles and Ultrasound.

Strategies for gene and nucleic acid delivery to skeletal muscles have been extensively explored to treat Duchenne muscular dystrophy (DMD) and other neuromuscular diseases. Of these, effective intravascular delivery of naked plasmid DNA (pDNA) and nucleic acids into muscles is an attractive approach, given the high capillary density in close contact with myofibers. We developed lipid-based nanobubbles (NBs) using polyethylene-glycol-modified liposomes and an echo-contrast gas and found that these NBs could improve tissue permeability by ultrasound (US)-induced cavitation. Herein, we delivered naked pDNA or antisense phosphorodiamidate morpholino oligomers (PMOs) into the regional hindlimb muscle via limb perfusion using NBs and US exposure. pDNA encoding the luciferase gene was injected with NBs via limb perfusion into normal mice with application of US. High luciferase activity was achieved in a wide area of the limb muscle. DMD model mice were administered PMOs, designed to skip the mutated exon 23 of the dystrophin gene, with NBs via intravenous limb perfusion, followed by US exposure. The number of dystrophin-positive fibers increased in the muscles of mdx mice. Combining NBs and US exposure, which can be widely delivered to the hind limb muscles via the limb vein, could be an effective therapeutic approach for DMD and other neuromuscular disorders.

Viability variation of T-cells under ultrasound exposure according to adhesion condition with bubbles.

PURPOSE: Although cellular immunotherapy is expected as a new cancer treatment, its therapeutic efficiency is limited in solid tumors, because most cells return to the bloodstream rather than adhere to the target site. Therefore, we are motivated to develop a technique to concentrate the cells in the blood flow using active control of bubble-surrounded cells under ultrasound exposure considering both aspects of cell controllability and viability. METHODS: We prepared a lipid bubble conjugating ligand to adhere to the surface of the T-cells. First, we evaluated the cell controllability by retaining the cells on a wall of an artificial blood vessel through continuous ultrasound exposure. Next, we investigated the cell viability under ultrasound exposure in a suspension with various bubble concentrations. RESULTS: We estimated the concentration of bubbles when the adhesion to the cell surface was saturated. Then, we evaluated the cell viability with various conditions of ultrasound exposure and bubble concentrations. However, it was confirmed that cell damage occurred under conditions that achieved proper control of the cells. Therefore, we exposed the cells to burst waves to reduce the applied ultrasound intensity. Consequently, the significant increase in cell viability was confirmed to be inversely proportional to the duty ratio. CONCLUSION: To retain cells on a vessel wall, determining the appropriate ultrasound condition including sound pressure and waveform is important to maintain cell viability.

Efficacy and Safety of Dietary Undenatured Type II Collagen on Joint and Motor Function in Healthy Volunteers: A Randomized, Double-Blind, Placebo-Controlled, Parallel-Group Study.

Prevalence of osteoarthritis (OA) is increasing alarmingly worldwide. Slowing down the progression of OA and diverse locomotive organ disorders is gaining interest in improving the quality of life (QOL) and extending healthy life-span. In a pilot study, intake of a small amount of undenatured type II collagen exhibited suppression of damage to the articular cartilage via oral immune tolerance. It also demonstrated improvement of knee and joint flexibility and mobility with continued intake of undenatured type II collagen (NEXT-II®) derived from chicken sternum cartilage. This randomized, double-blind, placebo-controlled, parallel-group clinical investigation (RCT) evaluated the efficacy and safety of 12 weeks of regular intake of NEXT-II® on joint and motor function in healthy Japanese male and female participants (aged 20 to <75 years).Sixty-four participants were randomized to receive either NEXT-II® (undenatured type II collagen 3.2 mg/d) or placebo over a period of 12 consecutive weeks. Efficacy on joint and motor functions were evaluated measuring knee passive range of motion as the primary outcome; the Japan Knee Osteoarthritis Measure (JKOM), Visual Analog Scale (VAS) for knee discomfort, and motor functions (10-meter walking and stair-climbing test) as the secondary outcomes; and Japan Low back pain Evaluation Questionnaire (JLEQ) and VAS for lower back discomfort as the exploratory outcomes.Fifty-eight participants (placebo = 28; NEXT-II® group = 30) completed the study. In the assessment of knee passive range of motion, significant improvements in "flexion" and "flexible angle (range)" were observed in the NEXT-II® group at 4, 8, and 12 weeks of treatment. NEXT-II® induced significant improvements in JKOM, VAS for knee and lower back discomfort, 10-meter walking test, stair-climbing test, and JLEQ.Results demonstrate that undenatured type II collagen is safe and efficacious in improving knee flexibility and mobility, reducing knee and lower back pain, and enhancing motor function.

Perinatal hypoxia aggravates occlusive pulmonary vasculopathy in SU5416/hypoxia-treated rats later in life.

Pulmonary arterial hypertension (PAH) is a fatal disease, which is characterized by occlusive pulmonary vascular disease (PVD) in small pulmonary arteries. It remains unknown whether perinatal insults aggravate occlusive PVD later in life. We tested the hypothesis that perinatal hypoxia aggravates PVD and survival in rats. PVD was induced in rats with/without perinatal hypoxia (embryonic day 14 to postnatal day 3) by injecting SU5416 at 7 wk of age and subsequent exposure to hypoxia for 3 wk (SU5416/hypoxia). Hemodynamic and morphological analyses were performed in rats with/without perinatal hypoxia at 7 wk of age (baseline rats, n = 12) and at 15 wk of age in 4 groups of rats: SU5416/hypoxia or control rats with/without perinatal hypoxia (n = 40). Pulmonary artery smooth muscle cells (PASMCs) from the baseline rats with/without perinatal hypoxia were used to assess cell proliferation, inflammation, and genomic DNA methylation profile. Although perinatal hypoxia alone did not affect survival, physiological, or pathological parameters at baseline or at the end of the experimental period in controls, perinatal hypoxia decreased weight gain and survival rate and increased right ventricular systolic pressure, right ventricular hypertrophy, and indices of PVD in SU5416/hypoxia rats. Perinatal hypoxia alone accelerated the proliferation and inflammation of cultured PASMCs from baseline rats, which was associated with DNA methylation. In conclusion, we established the first fatal animal model of PAH with worsening hemodynamics and occlusive PVD elicited by perinatal hypoxia, which was associated with hyperproliferative, proinflammatory, and epigenetic changes in cultured PASMCs. These findings provide insights into the treatment and prevention of occlusive PVD.

Focused ultrasound/microbubbles-assisted BBB opening enhances LNP-mediated mRNA delivery to brain.

Messenger RNA (mRNA) medicine has become a new therapeutic approach owing to the progress in mRNA delivery technology, especially with lipid nanoparticles (LNP). However, mRNA encapsulated-LNP (mRNA-LNP) cannot spontaneously cross the blood-brain barrier (BBB) which prevents the expression of foreign proteins in the brain. Microbubble-assisted focused ultrasound (FUS) BBB opening is an emerging technology that can transiently enhance BBB permeability. In this study, FUS/microbubble-assisted BBB opening was investigated for the intravenous delivery of mRNA-LNP to the brain. The intensity of FUS irradiation was optimized to 1.5 kW/cm2, at which BBB opening occurred efficiently without hemorrhage or edema. Exogenous protein (luciferase) expression by mRNA-LNP, specifically at the FUS-irradiated side of the brain, occurred only when FUS and microbubbles were applied. This exogenous protein expression was faster but shorter than that of plasmid DNA delivery. Furthermore, foreign protein expression was observed in the microglia, along with CD31-positive endothelial cells, whereas no expression was observed in astrocytes or neurons. These results support the addition of mRNA-LNP to the lineup of nanoparticles delivered by BBB opening.

Ultrasound and microbubble-mediated drug delivery and immunotherapy.

Ultrasound induces the oscillation and collapse of microbubbles such as those of an ultrasound contrast agent, where these behaviors generate mechanical and thermal effects on cells and tissues. These, in turn, induce biological responses in cells and tissues, such as cellular signaling, endocytosis, or cell death. These physiological effects have been used for therapeutic purposes. Most pharmaceutical agents need to pass through the blood vessel walls and reach the parenchyma cells to produce therapeutic effects in drug delivery. Therefore, the blood vessel walls act as an obstacle to drug delivery. The combination of ultrasound and microbubbles is a promising strategy to enhance vascular permeability, improving drug transport from blood to tissues. This combination has also been applied to gene and protein delivery, such as cytokines and antigens for immunotherapy. Immunotherapy, in particular, is an attractive technique for cancer treatment as it induces a cancer cell-specific response. However, sufficient anti-tumor effects have not been achieved with the conventional cancer immunotherapy. Recently, new therapies based on immunomodulation with immune checkpoint inhibitors have been reported. Immunomodulation can be regarded as a new strategy for cancer immunotherapy. It was also reported that mechanical and thermal effects induced by the combination of ultrasound and microbubbles could suppress tumor growth by promoting the cancer-immunity cycle via immunomodulation in the tumor microenvironment. In this review, we provide an overview of the application of ultrasound and microbubble combination for drug delivery and activation of the immune system in the microenvironment of tumor tissue.

CRISPR-mediated Bmpr2 point mutation exacerbates late pulmonary vasculopathy and reduces survival in rats with experimental pulmonary hypertension.

BACKGROUND: Patients with pulmonary arterial hypertension (PAH) carrying bone morphogenetic protein receptor type 2 (Bmpr2) mutations present earlier with severe hemodynamic compromise and have poorer survival outcomes than those without mutation. The mechanism underlying the worsening clinical phenotype of PAH with Bmpr2 mutations has been largely unaddressed in rat models of pulmonary hypertension (PH) because of the difficulty in reproducing progressive PH in mice and genetic modification in rats. We tested whether a clinically-relevant Bmpr2 mutation affects the progressive features of monocrotaline (MCT) induced-PH in rats. METHODS: A monoallelic single nucleotide insertion in exon 1 of Bmpr2 (+/44insG) was generated in rats using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9, then PH, pulmonary vascular disease (PVD) and survival after MCT injection with or without a phosphodiesterase type 5 inhibitor, tadalafil, administration were assessed. RESULTS: The +/44insG rats had reduced BMPR2 signalling in the lungs compared with wild-type. PH and PVD assessed at 3-weeks after MCT injection were similar in wild-type and +/44insG rats. However, survival at 4-weeks after MCT injection was significantly reduced in +/44insG rats. Among the rats surviving at 4-weeks after MCT administration, +/44insG rats had increased weight ratio of right ventricle to left ventricle plus septum (RV/[LV + S]) and % medial wall thickness (MWT) in pulmonary arteries (PAs). Immunohistochemical analysis showed increased vessels with Ki67-positive cells in the lungs, decreased mature and increased immature smooth muscle cell phenotype markers in the PAs in +/44insG rats compared with wild-type at 3-weeks after MCT injection. Contraction of PA in response to prostaglandin-F2α and endothelin-1 were significantly reduced in the +/44insG rats. The +/44insG rats that had received tadalafil had a worse survival with a significant increase in RV/(LV + S), %MWT in distal PAs and RV myocardial fibrosis compared with wild-type. CONCLUSIONS: The present study demonstrates that the Bmpr2 mutation promotes dedifferentiation of PA smooth muscle cells, late PVD and RV myocardial fibrosis and adversely impacts both the natural and post-treatment courses of MCT-PH in rats with significant effects only in the late stages and warrants preclinical studies using this new genetic model to optimize treatment outcomes of heritable PAH.

Lung damage created by high tidal volume ventilation in rats with monocrotaline-induced pulmonary hypertension.

BACKGROUND: Rats with chronic hypoxia-induced non-inflammatory pulmonary hypertension (PH) are resistant to ventilator-induced lung injury. We investigated the effect of high tidal volume ventilation in another model of PH, monocrotaline (MCT)-induced PH, which is a type of inflammatory PH. METHODS: PH was induced in rats by subcutaneous injection with 60 mg/kg MCT. Normal control rats, rats at 2 weeks after MCT injection (MCT2), and rats at 3 weeks after MCT injection (MCT3) were ventilated with low tidal volume (LV, 6 mL/kg) or high tidal volume (HV, 35 mL/kg) for 2 h with room air without positive end-expiratory pressure. Arterial oxygen pressure (PaO2) and Evans blue dye (EBD) extravasation were measured. Hypertensive pulmonary vascular remodeling was assessed morphometrically by the percentage of muscularized peripheral pulmonary arteries (%Muscularization) and the media wall thickness to external diameter ratio, namely percentage medial wall thickness (%MWT). To assess inflammation, lung IκB protein and cytokine mRNA expression levels were assessed. RESULTS: Baseline mean pulmonary arterial pressure was significantly higher in MCT rats (normal, 15.4 ± 0.5 mmHg; MCT2, 23.7 ± 0.9; and MCT3, 34.5 ± 1.5). After 2-h ventilation, PaO2 was significantly lower in the HV groups compared with the LV groups in normal and MCT2 rats, but not in MCT3 rats. Impairment of oxygenation with HV was less in MCT3 rats compared with normal and MCT2 rats. Among the HV groups, MCT3 rats showed significantly lower levels of EBD extravasation than normal and MCT2 rats. HV significantly downregulated IκB protein expression in normal and MCT3 rats and increased IL-6, MCP-1, CXCL-1 (MIP-1), and IL-10 mRNA levels in MCT3 rats. %Muscularization, %MWT, and the expression of lung elastin were significantly higher in MCT3 rats than in normal and MCT2 rats. CONCLUSION: We found that HV-associated damage might be reduced in MCT-induced PH rats compared with normal rats. The results of this and earlier studies suggest that hypertensive pulmonary vascular structural changes might be protective against the occurrence of ventilator-induced lung injury, irrespective of the etiology of PH.

Clinical Characteristics of Mechanically Ventilated Patients Treated in Non-ICU Settings in a Rural Area of Japan.

Introduction Patients requiring mechanical ventilation (MV) are commonly managed in an intensive care unit (ICU); however, Japan is unique in that many patients are treated in non-ICU settings. The characteristics of these patients, nevertheless, are unknown. We sought to identify disease severity and MV settings of patients in non-ICU. Methods We retrospectively analyzed the clinical data of Kinan Hospital and Owase General Hospital, where there are no ICUs. Data for adult patients who required MV from January through December 2018 were collected. To find the characteristics of patients who have been decided to treat in non-ICU hospitals without early transferring, we analyzed patients who have been treated for more than three days in those hospitals. Results A total of 171 patients received MV; 29 patients were treated for more than three days. Of those, the mortality rate was 44.8% (13 patients). The median age was 80 years (range: 72-84 years). The mean Acute Physiologic Assessment and Chronic Health Evaluation II (APACHE II) score was 20.9 ± 8.1, and predicted mortality was 0.42 ± 0.25. Tidal volume per predicted body weight was 8.8 ± 2.1 mL/kg, and set inspiratory time was 1.6 ± 0.3 seconds. Conclusions We have first described the severity and the initial ventilator setting of MV patients treated for more than three days in non-ICU setting in Japan. The overall predicted mortality was 42%, and the average age of the patients was 80 years. Further research on wider areas and the comparison to the patients treated in ICUs are needed to identify the appropriateness of treating patients in non-ICU settings.

Intermittent pain in patients with chronic low back pain is associated with abnormalities in muscles and fascia.

We investigated the relationship between paravertebral muscles and perimuscular connective tissues of the thoracolumbar fascia region and the four types of pain in patients suffering from chronic low back pain. A total of 17 patients with chronic low back pain participated in this study. Ultrasound imaging method was used to measure the thickness and echogenicity of the paravertebral muscles and perimuscular connective tissues. The measurement site considered in this study was located lateral to the midpoint between L2-3 and L4-5 spines. In addition, age, gender, BMI, numerical rating scale and the short-form McGill pain questionnaire 2 (includes questions with respect to continuous pain, intermittent pain, neuropathic pain and affective descriptors) were used for assessment. Statistical analysis was performed using correlation analysis and multiple regression analysis. A significant association was observed between paravertebral muscle echogenicity at L2-3 and the numerical rating scale (r = 0.499), between paravertebral muscle echogenicity at L4-5 with numerical rating scale (r = 0.538) and intermittent pain (r = 0.594), and between perimuscular connective tissue thickness at L2-3 and numerical rating scale (r = 0.762). We observed that the factor influencing perimuscular connective tissue thickness at L2-3 and L4-5 was intermittent pain (ß = 0.513, ß = 0.597, respectively). It was also observed that some of the imaging findings were associated with age and BMI. In conclusion, we observed that paravertebral muscle echogenicity and perimuscular connective tissue thickness in patients with chronic low back pain were associated with pain, especially intermittent pain.

Ultrasound image-guided gene delivery using three-dimensional diagnostic ultrasound and lipid-based microbubbles.

Gene therapy is a promising technology for genetic and intractable diseases. Drug delivery carriers or systems for genes and nucleic acids have been studied to improve transfection efficiency and achieve sufficient therapeutic effects. Ultrasound (US) and microbubbles have also been combined for use in gene delivery. To establish a clinically effective gene delivery system, exposing the target tissues to US is important. The three-dimensional (3D) diagnostic probe can three-dimensionally scan the tissue with mechanical regulation, and homogenous US exposure to the targeted tissue can be expected. However, the feasibility of therapeutically applying 3D probes has not been evaluated, especially gene delivery. In this study, we evaluated the characteristics of a 3D probe and lipid-based microbubbles (LB) for gene delivery and determined whether the 3D probe in the diagnostic US device could be used for efficient gene delivery to the targeted tissue using a mouse model. The 3D probe RSP6-16 with LB delivered plasmid DNA (pDNA) to the kidney after systemic injection with luciferase activity similar to that of probes used in previously studies. No toxicity was observed after treatment and, therefore, the combined 3D probe and LB would deliver genes to targeted tissue safely and efficiently.

Development of an Antibody Delivery Method for Cancer Treatment by Combining Ultrasound with Therapeutic Antibody-Modified Nanobubbles Using Fc-Binding Polypeptide.

A key challenge in treating solid tumors is that the tumor microenvironment often inhibits the penetration of therapeutic antibodies into the tumor, leading to reduced therapeutic efficiency. It has been reported that the combination of ultrasound-responsive micro/nanobubble and therapeutic ultrasound (TUS) enhances the tissue permeability and increases the efficiency of delivery of macromolecular drugs to target tissues. In this study, to facilitate efficient therapeutic antibody delivery to tumors using this combination system, we developed therapeutic antibody-modified nanobubble (NBs) using an Fc-binding polypeptide that can quickly load antibodies to nanocarriers; since the polypeptide was derived from Protein G. TUS exposure to this Herceptin®-modified NBs (Her-NBs) was followed by evaluation of the antibody's own ADCC activity, resulting the retained activity. Moreover, the utility of combining therapeutic antibody-modified NBs and TUS exposure as an antibody delivery system for cancer therapy was assessed in vivo. The Her-NBs + TUS group had a higher inhibitory effect than the Herceptin and Her-NBs groups. Overall, these results suggest that the combination of therapeutic antibody-modified NBs and TUS exposure can enable efficient antibody drug delivery to tumors, while retaining the original antibody activity. Hence, this system has the potential to maximize the therapeutic effects in antibody therapy for solid cancers.

Model difference in the effect of cilostazol on the development of experimental pulmonary hypertension in rats.

BACKGROUND: Preventing pulmonary vascular remodeling is a key strategy for pulmonary hypertension (PH). Causes of PH include pulmonary vasoconstriction and inflammation. This study aimed to determine whether cilostazol (CLZ), a phosphodiesterase-3 inhibitor, prevents monocrotaline (MCT)- and chronic hypoxia (CH)-induced PH development in rats. METHODS: Fifty-one male Sprague-Dawley rats were fed rat chow with (0.3% CLZ) or without CLZ for 21 days after a single injection of MCT (60 mg/kg) or saline. Forty-eight rats were fed rat chow with and without CLZ for 14 days under ambient or hypobaric (air at 380 mmHg) CH exposure. The mean pulmonary artery pressure (mPAP), the right ventricle weight-to-left ventricle + septum weight ratio (RV/LV + S), percentages of muscularized peripheral pulmonary arteries (%Muscularization) and medial wall thickness of small muscular arteries (%MWT) were assessed. Levels of the endothelial nitric oxide synthase (eNOS), phosphorylated eNOS (peNOS), AKT, pAKT and IκB proteins in lung tissue were measured using Western blotting. Monocyte chemotactic protein (MCP)-1 mRNA in lung tissue was also assessed. RESULTS: mPAP [35.1 ± 1.7 mmHg (MCT) (n = 9) vs. 16.6 ± 0.7 (control) (n = 9) (P < 0.05); 29.1 ± 1.5 mmHg (CH) (n = 10) vs. 17.5 ± 0.5 (control) (n = 10) (P < 0.05)], RV/LV + S [0.40 ± 0.01 (MCT) (n = 18) vs. 0.24 ± 0.01 (control) (n = 10) (P < 0.05); 0.41 ± 0.03 (CH) (n = 13) vs. 0.27 ± 0.06 (control) (n = 10) (P < 0.05)], and %Muscularization and %MWT were increased by MCT injection and CH exposure. CLZ significantly attenuated these changes in the MCT model [mPAP 25.1 ± 1.1 mmHg (n = 11) (P < 0.05), RV/LV + S 0.30 ± 0.01 (n = 14) (P < 0.05)]. In contrast, these CLZ effects were not observed in the CH model. Lung eNOS protein expression was unchanged in the MCT model and increased in the CH model. Lung protein expression of AKT, phosphorylated AKT, and IκB was downregulated by MCT, which was attenuated by CLZ; the CH model did not change these proteins. Lung MCP-1 mRNA levels were increased in MCT rats but not CH rats. CONCLUSIONS: We found model differences in the effect of CLZ on PH development. CLZ might exert a preventive effect on PH development in an inflammatory PH model but not in a vascular structural change model of PH preceded by vasoconstriction. Thus, the preventive effect of CLZ on PH development might depend on the PH etiology.

Enhanced Vascular Permeability by Microbubbles and Ultrasound in Drug Delivery.

Ultrasound and microbubbles, an ultrasound contrast agent, have recently increased attention to developing novel drug delivery systems. Ultrasound exposure can induce mechanical effects derived from microbubbles behaviors such as an expansion, contraction, and collapse depending on ultrasound conditions. These mechanical effects induce several biological effects, including enhancement of vascular permeability. For drug delivery, one promising approach is enhancing vascular permeability using ultrasound and microbubbles, resulting in improved drug transport to targeted tissues. This approach is applied to several tissues and drugs to cure diseases. This review describes the enhancement of vascular permeability by ultrasound and microbubbles and its therapeutic application, including our recent study. We also discuss the current situation of the field and its potential future perspectives.

Ternary Complexes of pDNA, Neuron-Binding Peptide, and PEGylated Polyethyleneimine for Brain Delivery with Nano-Bubbles and Ultrasound.

In brain-targeted delivery, the transport of drugs or genes across the blood-brain barrier (BBB) is a major obstacle. Recent reports found that focused ultrasound (FUS) with microbubbles enables transient BBB opening and improvement of drug or gene delivery. We previously developed nano-sized bubbles (NBs), which were prepared based on polyethylene glycol (PEG)-modified liposomes containing echo-contrast gas, and showed that our NBs with FUS could also induce BBB opening. The aim of this study was to enhance the efficiency of delivery of pDNA into neuronal cells following transportation across the BBB using neuron-binding peptides. This study used the RVG-R9 peptide, which is a chimeric peptide synthesized by peptides derived from rabies virus glycoprotein and nonamer arginine residues. The RVG peptide is known to interact specifically with the nicotinic acetylcholine receptor in neuronal cells. To enhance the stability of the RVG-R9/pDNA complex in vivo, PEGylated polyethyleneimine (PEG-PEI) was also used. The ternary complexes composed of RVG-R9, PEG-PEI, and pDNA could interact with mouse neuroblastoma cells and deliver pDNA into the cells. Furthermore, for the in vivo experiments using NBs and FUS, gene expression was observed in the FUS-exposed brain hemispheres. These results suggest that this systemic gene delivery system could be useful for gene delivery across the BBB.

Enhanced antitumor activity of combined lipid bubble ultrasound and anticancer drugs in gynecological cervical cancers.

Chemotherapy plays an important role in the treatment of patients with gynecological cancers. Delivering anticancer drugs effectively to tumor cells with just few side effects is key in cancer treatment. Lipid bubbles (LB) are compounds that increase the vascular permeability of the tumor under diagnostic ultrasound (US) exposure and enable the effective transport of drugs to tumor cells. The aim of our study was to establish a novel drug delivery technique for chemotherapy and to identify the most effective anticancer drugs for the bubble US-mediated drug delivery system (BUS-DDS) in gynecological cancer treatments. We constructed xenograft models using cervical cancer (HeLa) and uterine endometrial cancer (HEC1B) cell lines. Lipid bubbles were injected i.v., combined with either cisplatin (CDDP), pegylated liposomal doxorubicin (PLD), or bevacizumab, and US was applied to the tumor. We compared the enhanced chemotherapeutic effects of these drugs and determined the optimal drugs for BUS-DDS. Tumor volume reduction of HeLa and HEC1B xenografts following cisplatin treatment was significantly enhanced by BUS-DDS. Both CDDP and PLD significantly enhanced the antitumor effects of BUS-DDS in HeLa tumors; however, volume reduction by BUS-DDS was insignificant when combined with bevacizumab, a humanized anti-vascular endothelial growth factor mAb. The BUS-DDS did not cause any severe adverse events and significantly enhanced the antitumor effects of cytotoxic drugs. The effects of bevacizumab, which were not as dose-dependent as those of the two drugs used prior, were minimal. Our data suggest that BUS-DDS technology might help achieve "reinforced targeting" in the treatment of gynecological cancers.

Lipid bubbles combined with low-intensity ultrasound enhance the intratumoral accumulation and antitumor effect of pegylated liposomal doxorubicin in vivo.

Pegylated liposomal doxorubicin (PLD) is a representative nanomedicine that has improved tumor selectivity and safety profile. However, the therapeutic superiority of PLD over conventional doxorubicin has been reported to be insignificant in clinical medicine. Combination treatment with microbubbles and ultrasound (US) is a promising strategy for enhancing the antitumor effects of chemotherapeutics by improving drug delivery. Recently, several preclinical studies have shown the drug delivery potential of lipid bubbles (LBs), newly developed monolayer microbubbles, in combination with low-intensity US (LIUS). This study aimed to elucidate whether the combined use of LBs and LIUS enhanced the intratumoral accumulation and antitumor effect of PLD in syngeneic mouse tumor models. Contrast-enhanced US imaging using LBs showed a significant decrease in contrast enhancement after LIUS, indicating that LIUS exposure induced the destruction of LBs in the tumor tissue. A quantitative evaluation revealed that the combined use of LBs and LIUS improved the intratumoral accumulation of PLD. Furthermore, tumor growth was inhibited by combined treatment with PLD, LBs, and LIUS. Therefore, the combined use of LBs and LIUS enhanced the antitumor effect of PLD by increasing its accumulation in the tumor tissue. In conclusion, the present study provides important evidence that the combination of LBs and LIUS is an effective method for enhancing the intratumoral delivery and antitumor effect of PLD in vivo.

Inhibition of miR-96-5p in the mouse brain increases glutathione levels by altering NOVA1 expression.

Glutathione (GSH) is an important antioxidant that plays a critical role in neuroprotection. GSH depletion in neurons induces oxidative stress and thereby promotes neuronal damage, which in turn is regarded as a hallmark of the early stage of neurodegenerative diseases. The neuronal GSH level is mainly regulated by cysteine transporter EAAC1 and its inhibitor, GTRAP3-18. In this study, we found that the GTRAP3-18 level was increased by up-regulation of the microRNA miR-96-5p, which was found to decrease EAAC1 levels in our previous study. Since the 3'-UTR region of GTRAP3-18 lacks the consensus sequence for miR-96-5p, an unidentified protein should be responsible for the intermediate regulation of GTRAP3-18 expression by miR-96-5p. Here, we discovered that RNA-binding protein NOVA1 functions as an intermediate protein for GTRAP3-18 expression via miR-96-5p. Moreover, we show that intra-arterial injection of a miR-96-5p-inhibiting nucleic acid to living mice by a drug delivery system using microbubbles and ultrasound decreased the level of GTRAP3-18 via NOVA1 and increased the levels of EAAC1 and GSH in the dentate gyrus of the hippocampus. These findings suggest that the delivery of a miR-96-5p inhibitor to the brain would efficiently increase the neuroprotective activity by increasing GSH levels via EAAC1, GTRAP3-18 and NOVA1.

The Isoquinoline-Sulfonamide Compound H-1337 Attenuates SU5416/Hypoxia-Induced Pulmonary Arterial Hypertension in Rats.

Pulmonary arterial hypertension (PAH) is characterized by elevated pulmonary arterial pressure and right heart failure. Selective pulmonary vasodilators have improved the prognosis of PAH; however, they are not able to reverse pulmonary vascular remodeling. Therefore, a search for new treatment agents is required. H-1337 is an isoquinoline-sulfonamide compound that inhibits multiple serine/threonine kinases, including Rho-associated protein kinase (ROCK) and mammalian target of rapamycin (mTOR). Here, we investigated the effects of H-1337 on pulmonary hypertension and remodeling in the pulmonary vasculature and right ventricle in experimental PAH induced by SU5416 and hypoxia exposure. H-1337 and H-1337M1 exerted inhibitory effects on ROCK and Akt. H-1337 inhibited the phosphorylation of myosin light chain and mTOR and suppressed the proliferation of smooth muscle cells in vitro. H-1337 treatment also suppressed the phosphorylation of myosin light chain and mTOR in the pulmonary vasculature and decreased right ventricular systolic pressure and the extent of occlusive pulmonary vascular lesions. Furthermore, H-1337 suppressed aggravation of right ventricle hypertrophy. In conclusion, our data demonstrated that inhibition of ROCK and mTOR pathways with H-1337 suppressed the progression of pulmonary vascular remodeling, pulmonary hypertension, and right ventricular remodeling.