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.

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.

Scale-up production, characterization and toxicity of a freeze-dried lipid-stabilized microbubble formulation for ultrasound imaging and therapy.

Microbubble formulations have a long history for enhancement of ultrasound (US) imaging and recently also for therapeutic applications. Previously, a series of freeze-dried bubble formulations based on the lipids DSPC and DSPG were developed. Here, we have attempted to scale-up the production process for future more extensive studies. Bubbles were prepared by homogenization of a lipid dispersion in a perfluoropropane atmosphere in a medium size (300-500 mL) homogenizer and then freeze-dried for better storage stability. In total, 300 freeze-dried vials were prepared. The properties of the bubbles were similar to those previously prepared on a lab scale with the difference that they were slightly larger and also had a better stability. The re-entrapped gas concentration after re-constituted freeze-dried bubbles was 9.4 µL/µmol lipid. The re-entrapped rate was 72.3% of fresh bubble before freeze-drying (13.0 µL/µmol lipid). The half-life of US imaging signal of the re-constituted freeze-dried bubbles in water in vitro was shorter than that of the fresh bubbles (2.7 min vs. 3.3 min). A leak of Evans Blue, that binds to albumin, from mouse ear blood vessel was observed after combination of bubble and US irradiation of 1 MHz for 1 min. As a result of bubble vibration by US irradiation, vascular endothelial cell bond opened and Evans Blue leaked. Toxicity of bubble was tested in rats. No toxicity was found after a single injection in the dose range tested. No serious toxicity was seen after repeated injections (one daily injection during 15 days).

Development and evaluation of stability and ultrasound response of DSPC-DPSG-based freeze-dried microbubbles.

It is known that Phosphatidyl choline-Phosphatidyl glycerol mixtures can be used for liposome formulations, making them less leaky than liposomes with only one lipid. We hypothesized that this might also be the case for bubbles, which can be used as ultrasound (US) contrast agents. Therefore, we have compared a series of mixed distearoyl phosphatidylcholine-distearoyl phosphatidylglycerol (DSPC-DPSG) bubbles and with bubbles containing either DSPC or DSPG (and distearoyl ethanolamine-polyethyleneglycol 2000, DSPE-PEG2k). Here, we describe the development, examination of stability in vitro and attenuation of broad frequency US pulses. Novel lipid-stabilized freeze-dried formulations for US applications, using the phospholipids DSPC, DSPG, and PEGylated DSPE-PEG2k and perfluoropropane gas were developed. It was found that the bubbles could effectively be preserved by freeze-drying and then re-constituted by addition of water. Average bubble sizes were around 2 µm for all bubbles after re-constitution. Bubble stability was assessed by evaluating the decay of the US backscattering signal in vitro. Bubbles containing DSPG were more stable than bubbles with only DSPC. The composition DSPC:DSPG:DSPE-PEG2k 30:60:10 (molar ratio) was the most stable with an effective half-life of 9.12 min, compared to bubbles without DSPG, which had half-life of 2.05 min. Bubble attenuation of US depended highly on the compositions. Bubbles without DSPG had the highest attenuation indicating higher oscillation the most but were also destroyed by higher energy US. No bubbles with DSPG showed any indication of destruction but all had increased attenuations to varying degrees, DSPC:DSPG:DSPE-PEG2k 45:45:10 showed the least attenuation.

Tumor growth suppression by the combination of nanobubbles and ultrasound.

We previously developed novel liposomal nanobubbles (Bubble liposomes [BL]) that oscillate and collapse in an ultrasound field, generating heat and shock waves. We aimed to investigate the feasibility of cancer therapy using the combination of BL and ultrasound. In addition, we investigated the anti-tumor mechanism of this cancer therapy. Colon-26 cells were inoculated into the flank of BALB/c mice to induce tumors. After 8 days, BL or saline was intratumorally injected, followed by transdermal ultrasound exposure of tumor tissue (1 MHz, 0-4 W/cm2 , 2 min). The anti-tumor effects were evaluated by histology (necrosis) and tumor growth. In vivo cell depletion assays were performed to identify the immune cells responsible for anti-tumor effects. Tumor temperatures were significantly higher when treated with BL + ultrasound than ultrasound alone. Intratumoral BL caused extensive tissue necrosis at 3-4 W/cm2 of ultrasound exposure. In addition, BL + ultrasound significantly suppressed tumor growth at 2-4 W/cm2 . In vivo depletion of CD8+ T cells (not NK or CD4+ T cells) completely blocked the effect of BL + ultrasound on tumor growth. These data suggest that CD8+ T cells play a critical role in tumor growth suppression. Finally, we concluded that BL + ultrasound, which can prime the anti-tumor cellular immune system, may be an effective hyperthermia strategy for cancer treatment.

Lipid Nanoparticle with 1,2-Di-O-octadecenyl-3-trimethylammonium-propane as a Component Lipid Confers Potent Responses of Th1 Cells and Antibody against Vaccine Antigen.

Adjuvants are effective tools to enhance vaccine efficacy and control the type of immune responses such as antibody and T helper 1 (Th1)- or Th2-type responses. Several studies suggest that interferon (IFN)-γ-producing Th1 cells play a significant role against infections caused by intracellular bacteria and viruses; however, only a few adjuvants can induce a strong Th1-type immune response. Recently, several studies have shown that lipid nanoparticles (LNPs) can be used as vaccine adjuvants and that each LNP has a different adjuvant activity. In this study, we screened LNPs to develop an adjuvant that can induce Th1 cells and antibodies using a conventional influenza split vaccine (SV) as an antigen in mice. We observed that LNP with 1,2-di-O-octadecenyl-3-trimethylammonium-propane (DOTMA) as a component lipid (DOTMA-LNP) elicited robust SV-specific IgG1 and IgG2 responses compared with SV alone in mice and was as efficient as SV adjuvanted with other adjuvants in mice. Furthermore, DOTMA-LNPs induced robust IFN-γ-producing Th1 cells without inflammatory responses compared to those of other adjuvants, which conferred strong cross-protection in mice. We also demonstrated the high versatility of DOTMA-LNP as a Th1 cell-inducing vaccine adjuvant using vaccine antigens derived from severe acute respiratory syndrome coronavirus 2 and Streptococcus pneumoniae. Our findings suggest the potential of DOTMA-LNP as a safe and effective Th1 cell-inducing adjuvant and show that LNP formulations are potentially potent adjuvants to enhance the effectiveness of other subunit vaccines.

Ultrasound-mediated gene delivery systems by AG73-modified Bubble liposomes.

Targeted gene delivery to neovascular vessels in tumors is considered a promising strategy for cancer therapy. We previously reported that "Bubble liposomes" (BLs), which are ultrasound (US) imaging gas-encapsulating liposomes, were suitable for US imaging and gene delivery. When BLs are exposed to US, the bubble is destroyed, creating a jet stream by cavitation, and resulting in the instantaneous ejection of extracellular plasmid DNA (pDNA) or other nucleic acids into the cytosol. We developed AG73 peptide-modified Bubble liposomes (AG73-BL) as a targeted US contrast agent, which was designed to attach to neovascular tumor vessels and to allow specific US detection of angiogenesis (Negishi et al., Biomaterials 2013, 34, 501-507). In this study, to evaluate the effectiveness of AG73-BL as a gene delivery tool for neovascular vessels, we examined the gene transfection efficiency of AG73-BL with US exposure in primary human endothelial cells (HUVEC). The transfection efficiency was significantly enhanced if the AG73-BL attached to the HUVEC was exposed to US compared to the BL-modified with no peptide or scrambled peptide. In addition, the cell viability was greater than 80% after transfection with AG73-BL. These results suggested that after the destruction of the AG73-BL with US exposure, a cavitation could be effectively induced by the US exposure against AG73-BL binding to the cell surface of the HUVEC, and the subsequent gene delivery into cells could be enhanced. Thus, AG73-BL may be useful for gene delivery as well as for US imaging of neovascular vessels.

Bubble liposomes and ultrasound enhance the antitumor effects of AG73 liposomes encapsulating antitumor agents.

Encapsulating anticancer drugs in liposomes improves their therapeutic window by enhancing antitumor efficacy and reducing side effects. To devise more effective liposomal formulations for antitumor therapy, many research groups have tried to develop tumor-targeting liposomes with enhanced drug release. Previously, we developed doxorubicin (Dox)-encapsulated AG73 peptide-modified liposomes (AG73-Dox), which targeted cancer and endothelial cells, and ultrasound (US) imaging gas-entrapping liposomes, called "Bubble liposomes" (BLs). In this study, to enhance the antitumor effect of AG73-Dox, we combined AG73-Dox with BLs and US. First, to determine whether the addition of BLs and application of US could enhance the cytotoxicity of AG73-Dox, we evaluated the cytotoxicity of the combination of AG73-Dox with BLs and US. BLs and US enhanced cytotoxicity of AG73-Dox more than they enhanced nontargeted Dox-encapsulated liposomes. Next, we examined the intracellular behavior of Dox after treatment with BLs and US. The combination of AG73-Dox with BLs and US did not enhance cellular uptake of Dox, but it did promote drug release in the cytoplasm. To further elucidate the release of Dox in the cytoplasm, we blocked cellular uptake via endosomes at a low temperature. As a result, BLs and US did not have an enhanced drug-release effect until AG73-Dox was taken up into cells. Thus, the combination of AG73-Dox with BLs and US may be useful for cancer therapy as a dual-function drug delivery system with targeted and controlled release.

[Development of Microbubbles for Theranostics and Its Application in Brain Targeted Drug Delivery].

Theranostics, a new medical term that combines therapeutics and diagnostics is considered an ideal system for medical care. Ultrasound is considered one of the most reasonable energies for the development of theranostics. Additionally, microbubbles, which are ultrasound contrast agents, have received considerable attention for their effectiveness in diagnosis and therapy. Microbubbles are composed of an inner gas and an outer shell composed of proteins or phospholipids. Under ultrasound exposure, the oscillation or collapse of microbubbles is induced depending on the intensity of the ultrasound. These mechanical effects are important for imaging, drug delivery, and ablation therapies. Therefore, it is essential that microbubbles reach the targeted site and induce mechanical effects to achieve effective and efficient diagnosis and therapy. We have previously developed novel microbubbles with high stability by optimizing the outer shell composition. Recently, microbubbles containing distearoylphosphatidyl glycerol showed high stability and prolonged circulation in the blood. These novel microbubbles may be useful for diagnosis and therapy. The combination of microbubbles and ultrasound has received considerable attention for brain-targeted drug delivery applications. We examined whether microbubbles can be used for brain-targeted drug delivery and evaluated the effect of the encapsulated gas on drug delivery. Thus, novel microbubbles combined with ultrasound can deliver molecules to the brain. Microbubbles containing perfluoropropane or perfluorobutane could efficiently deliver molecules to the brain. The novel microbubbles have long-circulating properties in the blood and could deliver molecules to the brain. The combination of novel microbubbles and ultrasound would contribute to the development of efficient thranostic systems.

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.

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.

Involvement of Ca²âº and ATP in enhanced gene delivery by bubble liposomes and ultrasound exposure.

Recently, we reported the accelerated gene transfection efficiency of laminin-derived AG73-peptide-labeled polyethylene glycol-modified liposomes (AG73-PEG liposomes) and cell penetrating TAT-peptide labeled PEG liposomes using PEG-modified liposomes, which trap echo-contrast gas, "Bubble liposomes" (BLs), and ultrasound (US) exposure. BLs and US exposure were reported to enhance the endosomal escape of AG73-PEG liposomes, thereby leading to increased gene expression. However, the mechanism behind the effect of BLs and US exposure on endosomes is not well understood. US exposure was reported to induce an influx of calcium ions (Ca²âº) by enhancing permeability of the cell membrane. Therefore, we examined the effect of Ca²âº on the endosomal escape and transfection efficiency of AG73-PEG liposomes, which were previously enhanced by BLs and US exposure. For cells treated with EGTA, the endosomal escape and gene expression of AG73-PEG liposomes were not enhanced by BLs and US exposure. Similarly, transfection efficiency of the AG73-PEG liposomes in ATP-depleted cells was not enhanced. Our results suggest that Ca²âº and ATP are necessary for the enhanced endosomal escape and gene expression of AG73-PEG liposomes by BLs and US exposure. These findings may contribute to the development of useful techniques to improve endosomal escape and achieve efficient gene transfection.

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.

Feasibility study of novel nanoparticles derived from Glycyrrhizae radix as vaccine adjuvant for cancer immunotherapy.

Aims: The feasibility of using nanoparticles derived from Glycyrrhizae radix extract (Glycyrrhiza NPs) as a vaccine adjuvant for cancer immunotherapy was evaluated. Methods: C57BL/6J mice were immunized with ovalbumin (OVA) and Glycyrrhiza NPs. After immunization, splenocytes were incubated with the H-2Kb epitope peptide of OVA (SL8) and the production of IFN-γ was evaluated. Moreover, an OVA-expressing lymphoma cell line (E.G7-OVA cells) was inoculated into mice after immunization to evaluate the antitumor effect. Results: The immunization of OVA with Glycyrrhiza NPs induced IFN-γ production and completely rejected E.G7-OVA cells. Conclusion: Glycyrrhiza NPs could prime antigen-specific CD8+ T-cells resulting in antitumor effects. Therefore, Glycyrrhiza NPs can be an effective vaccine adjuvant for cancer immunotherapy.

Glycyrrhizae radix is a medical plant that contains anti-inflammatory compounds such as glycyrrhizin. Nanoparticles (NPs) derived from Glycyrrhizae radix extract induced the production of proinflammatory cytokines. Therefore, these NPs could be used as a vaccine adjuvant. Here, a feasibility study on the use of Glycyrrhiza NPs as a vaccine adjuvant in cancer immunotherapy is reported. T-cell responses and antitumor effects were evaluated after the immunization of ovalbumin (OVA) with Glycyrrhiza NPs. The immunization of OVA with Glycyrrhiza NPs effectively induced OVA-specific T-cells and completely rejected OVA-expressing tumor cells. Therefore, Glycyrrhiza NPs could induce antitumor immunity and be an effective vaccine adjuvant in cancer immunotherapy.

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 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.

Ultrasound-directed enzyme-prodrug therapy (UDEPT) using self-immolative doxorubicin derivatives.

Background: Enzyme-activatable prodrugs are extensively employed in oncology and beyond. Because enzyme concentrations and their (sub)cellular compartmentalization are highly heterogeneous in different tumor types and patients, we propose ultrasound-directed enzyme-prodrug therapy (UDEPT) as a means to increase enzyme access and availability for prodrug activation locally. Methods: We synthesized ß-glucuronidase-sensitive self-immolative doxorubicin prodrugs with different spacer lengths between the active drug moiety and the capping group. We evaluated drug conversion, uptake and cytotoxicity in the presence and absence of the activating enzyme ß-glucuronidase. To trigger the cell release of ß-glucuronidase, we used high-intensity focused ultrasound to aid in the conversion of the prodrugs into their active counterparts. Results: More efficient enzymatic activation was observed for self-immolative prodrugs with more than one aromatic unit in the spacer. In the absence of ß-glucuronidase, the prodrugs showed significantly reduced cellular uptake and cytotoxicity compared to the parent drug. High-intensity focused ultrasound-induced mechanical destruction of cancer cells resulted in release of intact ß-glucuronidase, which activated the prodrugs, restored their cytotoxicity and induced immunogenic cell death. Conclusion: These findings shed new light on prodrug design and activation, and they contribute to novel UDEPT-based mechanochemical combination therapies for the treatment of cancer.

Bubble liposomes and ultrasound promoted endosomal escape of TAT-PEG liposomes as gene delivery carriers.

We have previously developed laminin-derived AG73 peptide-labeled poly(ethylene glycol)-modified liposomes (AG73-PEG liposomes) for selective cancer gene therapy and reported that Bubble liposomes (BLs) and ultrasound (US) exposure could accelerate the endosomal escape of AG73-PEG liposomes, leading to the enhancement of transfection efficiency; however, it is still unclear whether BLs and US exposure can also enhance the transfection efficiency of other vectors. We therefore assessed the effect of BLs and US exposure on the gene transfection efficiency of trans-activating transcriptor (TAT) peptide modified PEG liposomes. Although TAT-PEG liposomes were efficiently internalized into cells, the efficacy of endosomal escape was insufficient. The transfection efficiencies of TAT-PEG liposomes were enhanced by about 30-fold when BLs and US exposure were used. We also confirmed that BLs and US exposure could not enhance the direct transportation of TAT-PEG liposomes into cells. Confocal microscopy showed that BLs and US exposure promoted endosomal escape of TAT-PEG liposomes. Our results suggested that BLs and US exposure could enhance transfection efficiency by promoting endosomal escape, which was independent of modified molecules of carriers. Thus, BLs and US exposure can be a useful tool to achieve efficient gene transfection by improving endosomal escape of various carriers.

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.