Intracranial Gene Delivery Mediated by Albumin-Based Nanobubbles and Low-Frequency Ultrasound.

Research in the field of high-intensity focused ultrasound (HIFU) for intracranial gene therapy has greatly progressed over the years. However, limitations of conventional HIFU still remain. That is, genes are required to cross the blood-brain barrier (BBB) in order to reach the neurological disordered lesion. In this study, we introduce a novel direct intracranial gene delivery method, bypassing the BBB using human serum albumin-based nanobubbles (NBs) injected through a less invasive intrathecal route via lumbar puncture, followed by intracranial irradiation with low-frequency ultrasound (LoFreqUS). Focusing on both plasmid DNA (pDNA) and messenger RNA (mRNA), our approach utilizes LoFreqUS for deeper tissue acoustic penetration and enhancing gene transfer efficiency. This drug delivery method could be dubbed as the "Spinal Back-Door Approach", an alternative to the "front door" BBB opening method. Experiments showed that NBs effectively responded to LoFreqUS, significantly improving gene transfer in vitro using U-87 MG cell lines. In vivo experiments in mice demonstrated significantly increased gene expression with pDNA; however, we were unable to obtain conclusive results using mRNA. This novel technique, combining albumin-based NBs and LoFreqUS offers a promising, efficient, targeted, and non-invasive solution for central nervous system gene therapy, potentially transforming the treatment landscape for neurological disorders.

Efficient mRNA Delivery with Lyophilized Human Serum Albumin-Based Nanobubbles.

In this study, we developed an efficient mRNA delivery vehicle by optimizing a lyophilization method for preserving human serum albumin-based nanobubbles (HSA-NBs), bypassing the need for artificial stabilizers. The morphology of the lyophilized material was verified using scanning electron microscopy, and the concentration, size, and mass of regenerated HSA-NBs were verified using flow cytometry, nanoparticle tracking analysis, and resonance mass measurements, and compared to those before lyophilization. The study also evaluated the response of HSA-NBs to 1 MHz ultrasound irradiation and their ultrasound (US) contrast effect. The functionality of the regenerated HSA-NBs was confirmed by an increased expression of intracellularly transferred Gluc mRNA, with increasing intensity of US irradiation. The results indicated that HSA-NBs retained their structural and functional integrity markedly, post-lyophilization. These findings support the potential of lyophilized HSA-NBs, as efficient imaging, and drug delivery systems for various medical applications.

Influence of Nanobubble Size Distribution on Ultrasound-Mediated Plasmid DNA and Messenger RNA Gene Delivery.

The use of nanobubbles (NBs) for ultrasound-mediated gene therapy has recently attracted much attention. However, few studies have evaluated the effect of different NB size distribution to the efficiency of gene delivery into cells. In this study, various size of albumin stabilized sub-micron bubbles were examined in an in vitro ultrasound (1 MHz) irradiation setup in the aim to compare and optimize gene transfer efficiency. Results with pDNA showed that gene transfer efficiency in the presence of NB size of 254.7 ± 3.8 nm was 2.5 fold greater than those with 187.3 ± 4.8 nm. Similarly, carrier-free mRNA transfer efficiency increased in the same conditions. It is suggested that NB size greater than 200 nm contributed more to the delivery of genes into the cytoplasm with ultrasound. Although further experiments are needed to understand the underlying mechanism for this phenomenon, the present results offer valuable information in optimizing of NB for future ultrasound-mediate gene therapy.

Nanobubble Mediated Gene Delivery in Conjunction With a Hand-Held Ultrasound Scanner.

Recent research has revealed that nanobubbles (NBs) can be an effective tool for gene transfection in conjunction with therapeutic ultrasound (US). However, an approach to apply commercially available hand-held diagnostic US scanners for this purpose has not been evaluated as of now. In the present study, we first compared in vitro, the efficiency of gene transfer (pCMV-Luciferase) with lipid-based and albumin-based NBs irradiated by therapeutic US (1MHz, 5.0 W/cm2) in oral squamous carcinoma cell line HSC-2. Secondly, we similarly examined if gene transfer in mice is possible using a clinical hand-held US scanner (2.3MHz, MI 1.0). Results showed that lipid-based NBs induced more gene transfection compared to albumin-based NBs, in vitro. Furthermore, significant gene transfer was also obtained in mice liver with lipid-based NBs. Sub-micro sized bubbles proved to be a powerful gene transfer reagent in combination with conventional hand-held ultrasonic diagnostic device.

Echographic and physical characterization of albumin-stabilized nanobubbles.

There has been increasing interest in using nanobubbles (NBs) for ultrasound mediated drug delivery as well as for ultrasound imaging. Albumin NBs are especially attractive for its potential of becoming a versatile platform for drug carriers and molecular targeted therapy agents. However, physical characterization of NBs is generally considered to be difficult due to various technical issues, such as concentration limitations, nanoparticle contamination, etc. In the present study, we measured the size distribution, concentration and weight density of albumin stabilized NBs by means of multiple nanoscale measurement modalities. Laser nanoparticle tracking analysis, multicolor flow cytometry, resonance mass evaluation showed consistent measurement results of the NBs with low mass weight density and diameter size ranging from 100 nm to 400 nm. Furthermore, the NB solution showed excellent images by high frequency ultrasound (30-50 MHz) in flow model acoustic phantoms. The NBs also induced acute cell disruption by low intensity ultrasound (0.8 W/cm2) irradiation. We successfully fabricated and characterized albumin stabilized NBs which could serve as an effective platform for future theranositic agents.

Enhanced cell killing and apoptosis of oral squamous cell carcinoma cells with ultrasound in combination with cetuximab coated albumin microbubbles.

Targeted microbubbles have the potential to be used for ultrasound (US) therapy and diagnosis of various cancers. In the present study, US was irradiated to oral squamous cell carcinoma cells (HSC-2) in the presence of cetuximab-coated albumin microbubbles (CCAM). Cell killing rate with US treatment at 0.9 W/cm2 and 1.0 W/cm2 in the presence of CCAM was greater compared to non-targeted albumin microbubbles (p < .05). On the other hand, selective cell killing was not observed in human myelomonocytic lymphoma cell line (U937) that had no affinity to cetuximab. Furthermore, US irradiation in the presence of CCAM showed a fivefold increase of cell apoptotic rate for HSC-2 cells (21.0 ± 3.8%) as compared to U937 cells (4.0 ± 0.8%). Time-signal intensity curve in a tissue phantom demonstrated clear visualisation of CCAM with conventional US imaging device. Our experiment verifies the hypothesis that CCAM was selective to HSC-2 cells and may be applied as a novel therapeutic/diagnostic microbubble for oral squamous cell carcinoma.

Acute effects of sono-activated photocatalytic titanium dioxide nanoparticles on oral squamous cell carcinoma.

Sonodynamic therapy (SDT) is a new treatment modality using ultrasound to activate certain chemical sensitizers for cancer therapy. In this study, effects of high intensity focused ultrasound (HIFU) combined with photocatalytic titanium dioxide (TiO2) nanoparticles on human oral squamous cell line HSC-2 were investigated. Viability of HSC-2 cells after 0, 0.1, 1, or 3s of HIFU irradiation with 20, 32, 55 and 73Wcm(-2) intensities in the presence or absence of TiO2 was measured immediately after the exposures in vitro. Immediate effects of HIFU (3s, 73Wcm(-2)) combined with TiO2 on solid tumors were also examined by histological study. Cytotoxic effect of HIFU+TiO2in vitro was significantly higher than that of TiO2 or HIFU alone with the tendency to increase for higher HIFU intensity, duration, and TiO2 concentration in the suspension. In vivo results showed significant necrosis and tissue damage in HIFU and HIFU+TiO2 treated samples. However, penetration of TiO2 nanoparticles into the cell cytoplasm was only observed in HIFU+TiO2 treated tissues. In this study, our findings provide a rational basis for the development of an effective HIFU based sonodynamic activation method. This approach offers an attractive non-invasive therapy technique for oral cancer in future.

Enhanced mechanical damage to in vitro cancer cells by high-intensity-focused ultrasound in the presence of microbubbles and titanium dioxide.

PURPOSE: To evaluate in vitro the feasibility of therapeutic high-intensity-focused ultrasound (HIFU) combined with microbubbles and titanium dioxide (TiO2). METHODS: Oral squamous cell carcinoma cells (HSC-2) were sonicated using a HIFU transducer with a resonant frequency of 3.5 MHz, 30 mm in diameter, and focal length of 50 mm. The ultrasound intensity was 210 W/cm(2), and two pulses (0.5 s each) were sonicated for each cell sample (9 × 10(4) cells per well). Immediately after HIFU, the viable cells were measured by an automated cell counter. The survival rate was measured in the presence of microbubbles (Sonazoid) and peroxo titania-silica (R-P-TS) or anatase titania-silica (R-A-TS) TiO2. RESULTS: Cell viability immediately following sonication in the presence of TiO2 (R-A-TS) and TiO2 (R-P-TS) was 65.5 ± 0.7 and 59.4 ± 3.3 %, respectively. A marked decrease in cell viability was seen when microbubbles were added to the above cell conditions. Specifically, cell viability decreased to 14.0 ± 0.1 and 4.4 ± 0.9 % when microbubbles were added to samples containing TiO2 (R-A-TS) and TiO2 (R-P-TS), respectively. CONCLUSION: Immediate in vitro cell killing was observed with short pulsed duration HIFU sonication with a combination of microbubbles and TiO2. This finding suggests that TiO2 could have caused enhanced mechanical cell destruction by microbubbles.

A target-controlled infusion system with bispectral index monitoring of propofol sedation during endoscopic submucosal dissection.

BACKGROUND AND STUDY AIMS: Propofol administration via a target-controlled infusion system with bispectral index monitoring (BIS/TCI system) is expected to prevent complications from sedation during complex and long endoscopic procedures. We evaluated the feasibility of setting the BIS/TCI system for non-anesthesiologist administration of propofol (NAAP) during endoscopic submucosal dissection (ESD). PATIENTS AND METHODS: From May 2009 to February 2013, 250 patients with esophagogastric neoplasms were treated with ESD using the BIS/TCI system with NAAP. In the TCI system, the initial target blood concentration of propofol was set at 1.2 µg/mL. The titration speed of propofol was adjusted according to the BIS score and the movement of the patient. The BIS target level ranged from moderate to deep sedation, at which a stable BIS score between 60 and 80 was obtained. RESULTS: In 80.4 % of patients, it was possible to maintain stable sedation with a blood concentration of propofol of less than 1.6 µg/mL using TCI throughout the ESD procedure. The default setting for ideal blood concentration of propofol was 1.2 µg/mL, because the medians of the lower and upper bounds of blood concentration were 1.2 µg/mL (range 0.6 - 1.8 µg/mL) and 1.4 µg/mL (range 1.0 - 3.8 µg/mL), respectively. Although hypotension occurred in 27 patients (10.8 %), oxygen desaturation occurred in only nine patients (3.6 %), and severe desaturation in only two patients (0.8 %). CONCLUSIONS: Using our settings, it is possible for a non-anesthesiologist to maintain stable sedation during a lengthy endoscopic procedure through propofol sedation with a BIS/TCI system.

[A case of renal salt-wasting syndrome during chemotherapy for advanced gastric cancer].

A 66-year-old woman was admitted to our hospital with heartburn and liver dysfunction. She was diagnosed with advanced gastric cancer. After the initiation of chemotherapy with trastuzumab, capecitabine, and cisplatin, she developed hyponatremia and renal failure with renal salt-wasting syndrome (RSWS). She recovered from these conditions after infusion of hypertonic saline. A diagnosis of RSWS should be considered in patients with hyponatremia who receive cisplatin-based chemotherapy.

Ultrasound-mediated interferon ß gene transfection inhibits growth of malignant melanoma.

We investigated the effects of ultrasound-mediated transfection (sonotransfection) of interferon ß (IFN-ß) gene on melanoma (C32) both in vitro and in vivo. C32 cells were sonotransfected with IFN-ß in vitro. Subcutaneous C32 tumors in mice were sonicated weekly immediately after intra-tumor injection with IFN-ß genes mixed with microbubbles. Successful sonotransfection with IFN-ß gene in vitro was confirmed by ELISA, which resulted in C32 growth inhibition. In vivo, the growth ratio of tumors transfected with IFN-ß gene was significantly lower than the other experimental groups. These results may lead to a new method of treatment against melanoma and other hard-to-treat cancers.

Synergistic inhibition of malignant melanoma proliferation by melphalan combined with ultrasound and microbubbles.

The cavitational effects of ultrasound (US) exposure induce transient pores on the cell membrane (sonoporation). Sonoporation have been applied in the field of cancer therapy by promoting delivery of extracellular molecules such as drugs and genes into cytoplasm. In addition, it is known that using US together with microbubbles (MB) elevates permeability of these agents. In this study, by applying the US-MB strategy for melanoma chemotherapy, we evaluated the antitumor effect of melphalan combined with US-MB on a melanoma cell line (C32) in vitro and in vivo. The in vitro cytotoxic effect of the melphalan with US-MB was greater than that of melphalan alone or melphalan in combination with US. In vivo experiments using xenografts, intratumoral injection of melphalan and MB with US exposure led to a greater degree of tumor regression than did the intratumoral injection of the melphalan alone or melphalan in combination with US. These results suggest that US-MB promotes the antitumor effect of melphalan by increasing delivery of molecules into cells and that this strategy may become an effective method of adjuvant therapy against malignant melanoma.

Ultrasound activation of TiO2 in melanoma tumors.

Sonodynamic therapy (SDT) is a new modality using ultrasound (US) to activate certain chemical sensitizers for cancer therapy. In this study, the effect of US combined with a nanoparticle titanium dioxide (TiO(2)) on melanoma cell was investigated in vitro and in vivo. Melanoma cells (C32) were irradiated with US in the presence and/or absence of TiO(2). Cell viability was measured immediately after US irradiation (1MHz, 0.5 and 1.0W/cm(2) for 10s). The effect of the combination of TiO(2) and US exposure (1MHz, 1.0W/cm(2), 2 min duration) on subcutaneously implanted C32 solid tumors in mice were investigated by measuring tumor volume regression. The cell viability was significantly decreased only after US irradiation in the presence of TiO(2). In vivo results showed significant inhibition of tumor growth in groups treated with TiO(2) and US. To our knowledge, this is the first report to demonstrate the cell killing effect of TiO(2) nanoparticles under the irradiation US in vitro and in vivo.

Synergistic effect of ultrasound and antibiotics against Chlamydia trachomatis-infected human epithelial cells in vitro.

To investigate whether or not the combined ultrasound and antibiotic treatment is effective against chlamydial infection, a new ultrasound exposure system was designed to treat chlamydia-infected cells. First, the minimum inhibitory concentrations of antibiotics against Chlamydia trachomatis were determined. Infected cultures were treated with antibiotics then sonicated at intensity of 0.15 or 0.44 W/cm(2) with or without Bubble liposomes. After 48 or 72 h after infection, chlamydial inclusions were stained and examined by fluorescence microscopy. The internalization of dextran-fluorescein conjugates by ultrasound irradiation with Bubble liposomes was observed by fluorescence microscopy. The results showed that application of nanobubble-enhanced ultrasound caused no significant effect on cell viability and chlamydial infectivity. However, Doxycycline (1/2 MIC) or CZX (1.0 µg/ml) in combination with nanobubble-enhanced ultrasound dramatically reduced the number of inclusions compared with that administered with antibiotics only. Bubble dose-dependent synergy was also observed. After ultrasound irradiation at intensity of 0.44 W/cm(2) on the presence of Bubble liposomes, 10% of HeLa cells were observed to have internalized the dextran molecules. This study suggests the possibility of using nanobubble-enhanced ultrasound to deliver antibiotic molecules into cells to eradiate intracellular bacteria, such as chlamydiae, without causing much damage to the cells itself.

Hypotonia-induced cell swelling enhances ultrasound-induced mechanical damage to cancer cells.

INTRODUCTION: It has been shown that killing of suspended cells by low-intensity ultrasound (0.08-0.11 W/cm(2)) can be enhanced by a mild non-lethal hypotonic (146 mOsm) medium. PURPOSE: In this study we wished to determine whether hypotonia-induced cell swelling of suspension cells was directly related to enhancement of ultrasound-mediated cell killing, and to verify whether similar effects could be observed on circulating and attached cells. METHODS: U937 cells under mild hypotonia were exposed to ultrasound for different times with real-time monitoring of cell size using a particle-size-distribution analyzer. To study the effect on attached cells, HeLa cells were exposed to ultrasound while under hypotonia in an in vivo-simulated set-up. RESULTS: The result showed that the enhanced cell killing (up to more than twice) was directly proportional to hypotonia-induced cell swelling. Similar membrane damage based on PI staining could be observed on HeLa cells treated with hypotonia. An in vivo-simulated circulating system also showed similar findings for hypotonia-enhanced ultrasound cell killing. CONCLUSION: These findings showed that mild hypotonia can be used to augment the effect of ultrasound in the treatment of cancers, particularly leukemia. The results showing that such enhancement is related to cell swelling could guide us toward proper timing of sonication while under hypotonic treatment.

Growth inhibition of neurofibroma by ultrasound-mediated interferon γ transfection.

PURPOSE: We have previously shown that ultrasound-mediated transfection (sonotransfection) can be optimized using a concept based on the ultrasound-induced apoptosis produced in our in vitro experiments. At optimized conditions, we have shown, using five cancer cell lines, that sonotransfection is superior to other conventional nonviral methods. Interferon gamma (IFN-γ) transfection using lipofection has been found to markedly inhibit the proliferation of neurofibroma cell lines. In this study, we investigated whether sonotransfection of IFN-γ to neurofibroma cell lines can suppress cell proliferation. METHODS: The ultrasound device used was the SonoPore KTAC-4000, which is capable of various acoustic settings. Ultrasound transducers at an oscillation frequency of 1.011 MHz were used; the potential ideal conditions were an intensity of 0.17 W/cm(2) at a burst frequency of 0.5 Hz, 25% duty factor, and 30-s sonication duration. Cells were assayed at 3 and 5 days after sonication. RESULTS: The transfection efficiency was found to be 12%. The ultrasound-treated cells were successfully transfected with IFN-γ genes as detected by enzyme-linked immunosorbent assay, and the cell growth ratio in the IFN-γ sonotransfection group tended to be lower than that in the other experimental groups. CONCLUSION: These results suggested that IFN-γ sonotransfection could potentially become a nonsurgical method for treating skin lesions such as neurofibromas.

Therapeutic potential of low-intensity ultrasound (part 2): biomolecular effects, sonotransfection, and sonopermeabilization.

Part one of this review focused on the thermal and mechanical effects of low-intensity ultrasound (US). In this second and final part of the review, we will focus on and discuss various aspects of low-intensity US, with emphasis on the biomolecular effects, US-mediated gene transfection (sonotransfection), and US-mediated permeabilization (sonopermeabilization). Sonotransfection of different cell lines in vitro and target tissues in vivo have been reported. Optimization experiments have been done and different mechanisms investigated. It has also been found that several genes can be up-regulated or down-regulated by sonication. As to the potential therapeutic applications, systemic or local sonotransfection might also be a safe and effective gene therapy method in effecting the cure of local and systemic disorders. Gene regulation of target cells may be utilized in modifying cellular response to a treatment, such as increasing the sensitivity of diseased cells while making normal cells resistant to the side effects of a treatment. Advances in sonodynamic therapy and drug sonopermeabilization also offer an ever-increasing array of therapeutic options for low-intensity US.