Toxicological effects of cationic nanobubbles on the liver and kidneys: Biomarkers for predicting the risk

Nanobubbles with acoustical activity are used as both diagnostic and therapeutic carriers for detecting and treating diseases. We aimed to prepare nanobubbles and assess toxic responses to them in the liver and kidneys. The cytotoxicity of nanobubbles was determined by examining the viability of liver (HepG2) and kidney (293T) cell lines after a 24-h treatment at various concentrations (0.01–2%). Toxic effects of different formulations were compared by determining functional markers such as γ-glutamyl transferase (γ-GT) and blood urea nitrogen (BUN) after intravenous administration of nanobubbles. Cationic nanobubbles caused concentration-dependent cytotoxicity against cultured cells with a more significant effect in the liver than in the kidneys. A significant reduction of viability was revealed at a concentration as low as 0.1%. Cational systems with soyaethyl morpholinium ethosulfate (SME) exhibited the greatest γ-GT level at 6-fold higher than the control. Immunohistochemistry detected liver fibrosis and inflammation with nanobubbles treatment, especially SME-containing ones at higher doses. According to plasma proteomic profiles, gelsolin and fetuin-B were significantly downregulated 3-fold in the high-dose SME-treated group. Transthyretin decreased by 6-fold in this group. The fibrinogen gamma chain expression was highly elevated. The results suggest that these protein biomarkers are sensitive for assessing the risk of nanobubble exposure. This study is the first to systematically evaluate the possible toxicity of nanobubbles in the liver and kidneys.     

Preparation and characterization of dextran nanobubbles for oxygen delivery

Dextran nanobubbles were prepared with a dextran shell and a perfluoropentan core in which oxygen was stored. To increase the stability polyvinylpirrolidone was also added to the formulation as stabilizing agent. Rhodamine B was used as fluorescent marker to obtain fluorescent nanobubbles. The nanobubble formulations showed sizes of about 500 nm, a negative surface charge and a good capacity of loading oxygen, no hemolytic activity or toxic effect on cell lines. The fluorescent labelled nanobubbles could be internalized in Vero cells. Oxygen-filled nanobubbles were able to release oxygen in different hypoxic solutions at different time after their preparation in in vitro experiments. The oxygen release kinetics could be enhanced after nanobubble insonation with ultrasound at 2.5 MHz. The oxygen-filled nanobubble formulations might be proposed for therapeutic applications in various diseases.     

Methotrexate-loaded PLGA nanobubbles for ultrasound imaging and Synergistic Targeted therapy of residual tumor during HIFU ablation

High intensity focused ultrasound (HIFU) has attracted the great attention in tumor ablation due to its non-invasive, efficient and economic features. However, HIFU ablation has its intrinsic limitations for removing the residual tumor cells, thus the tumor recurrence and metastasis cannot be avoided in this case. Herein, we developed a multifunctional targeted poly(lactic-co-glycolic acid) (PLGA) nanobubbles (NBs), which not only function as an efficient ultrasound contrast agent for tumor imaging, but also a targeted anticancer drug carrier and excellent synergistic agent for enhancing the therapeutic efficiency of HIFU ablation. Methotrexate (MTX)-loaded NBs were synthesized and filled with perfluorocarbon gas subsequently using a facile but general double emulsion evaporation method. The active tumor-targeting monoclonal anti-HLA-G antibodies (mAb_HLA-G) were further conjugated onto the surface of nanobubbles. The mAb_HLA-G/MTX/PLGA NBs could enhance the ultrasound imaging both in vitro and in vivo, and the targeting efficiency to HLA-G overexpressing JEG-3 cells has been demonstrated. The elaborately designed mAb_HLA-G/MTX/PLGA NBs can specifically target to the tumor cells both in vitro and in vivo, and their blood circulation time in vivo was much longer than non-targeted MTX/PLGA NBs. Further therapeutic evaluations showed that the targeted NBs as a synergistic agent can significantly improve the efficiency of HIFU ablation by changing the acoustic environment, and the focused ultrasound can promote the on-demand MTX release both in vitro and in vivo. The in vivo histopathology test and immunohistochemical analysis showed that the mAb_HLA-G/MTX/PLGA NBs plus HIFU group presented most serious coagulative necrosis, the lowest proliferation index and the highest apoptotic index. Therefore, the successful introduction of targeted mAb_HLA-G/MTX/PLGA NBs provides an excellent platform for the highly efficient, imaging-guided and non-invasive HIFU synergistic therapy of cancer with the supplementary functions of killing residual tumor cells and preventing tumor recurrence/metastasis.     

Micro- and nanobubbles: A versatile non-viral platform for gene delivery

Micro- and nanobubbles provide a promising non-viral strategy for ultrasound mediated gene delivery. Microbubbles are spherical gas-filled structures with a mean diameter of 1–8 μm, characterised by their core–shell composition and their ability to circulate in the bloodstream following intravenous injection. They undergo volumetric oscillations or acoustic cavitation when insonified by ultrasound and, most importantly, they are able to resonate at diagnostic frequencies. It is due to this behaviour that microbubbles are currently being used as ultrasound contrast agents, but their use in therapeutics is still under investigation. For example, microbubbles could play a role in enhancing gene delivery to cells: when combined with clinical ultrasound exposure, microbubbles are able to favour gene entry into cells by cavitation. Two different delivery strategies have been used to date: DNA can be co-administered with the microbubbles (i.e. the contrast agent) or ‘loaded’ in purposed-built bubble systems – indeed a number of different technological approaches have been proposed to associate genes within microbubble structures. Nanobubbles, bubbles with sizes in the nanometre order of magnitude, have also been developed with the aim of obtaining more efficient gene delivery systems. Their small sizes allow the possibility of extravasation from blood vessels into the surrounding tissues and ultrasound-targeted site-specific release with minimal invasiveness. In contrast, microbubbles, due to their larger sizes, are unable to extravasate, thus and their targeting capacity is limited to specific antigens present within the vascular lumen. This review provides an overview of the use of microbubbles as gene delivery systems, with a specific focus on recent research into the development of nanosystems. In particular, ultrasound delivery mechanisms, formulation parameters, gene-loading approaches and the advantages of nanometric systems will be described.     

Activated human neutrophil response to perfluorocarbon nanobubbles: oxygen-dependent and -independent cytotoxic responses

Nanobubbles, a type of nanoparticles with acoustically active properties, are being utilized as diagnostic and therapeutic nanoparticles to better understand, detect, and treat human diseases. The objective of this work was to prepare different nanobubble formulations and investigate their physicochemical characteristics and toxic responses to N-formyl-methionyl-leucyl-phenylalanine (fMLP)-activated human neutrophils. The nanobubbles were prepared using perfluoropentane and coconut oil as the respective core and shell, with soybean phosphatidylcholine (SPC) and/or cationic surfactants as the interfacial layers. The cytotoxic effect of the nanobubbles on neutrophils was determined by extracellular O₂⁻ release, intracellular reactive oxygen species (ROS), lactate dehydrogenase (LDH), and elastase release. Particle sizes of the nanobubbles with different percentages of perfluorocarbon, oil, and surfactants in ranged 186-432 nm. The nanobubbles were demonstrated to inhibit the generation of superoxide and intracellular ROS. The cytotoxicity of nanobubbles may be mainly associated with membrane damage, as indicated by the high LDH leakage. Systems with Forestall (FE), a cationic surfactant, or higher SPC contents exhibited the greatest LDH release by 3-fold compared to the control. The further addition of an oil component reduced the cytotoxicity induced by the nanobubbles. Exposure to most of the nanobubble formulations upregulated elastase release by activated neutrophils. Contrary to this result, stearylamine (SA)-containing systems slightly but significantly suppressed elastase release. FE and SA in a free form caused stronger responses by neutrophils than when they were incorporated into nanobubbles. In summary, exposure to nanobubbles resulted in a formulation-dependent toxicity toward human neutrophils that was associated with both oxygen-dependent and -independent pathways. Clinicians should therefore exercise caution when using nanobubbles in patients for diagnostic and drug targeting aims.     

对比观察脂质纳泡与微泡在高强度聚焦超声消融兔肝中的增效作用

目的观察正常兔肝中脂质纳泡与微泡对高强度聚焦超声(HIFU)消融效果的影响,探讨纳泡的应用价值。方法采用机械振荡法联合低速离心制备纳泡,观察和分析纳泡及微泡的形态、大小及分布。18只健康新西兰兔随机分为3组:HIFU+生理盐水组、HIFU+微泡组及HIFU+纳泡组,耳缘静脉注入各溶液15s后开始HIFU辐照,辐照功率为180 W,辐照时间5s,观察HIFU辐照前后回声变化,检测靶区组织的凝固性坏死体积以及微细结构变化,并作统计学分析。结果制备的脂质纳泡粒径均一,纳泡、微泡的平均粒径分别为(588.00±53.02)nm、(3058.00±545.20)nm;HIFU+微泡组与HIFU+纳泡组,靶区凝固性坏死体积[(124.26±16.72)mm3,(121.35±11.25)mm3]差异无统计学意义(P0.05),但均显著大于HIFU+生理盐水组(62.49±4.54)mm3(P均0.05);各组坏死组织微细结构均严重破坏。结论脂质纳泡具有与微泡相同的HIFU增效作用,为纳泡在HIFU技术中的深入研究提供实验依据。     

Tumor-penetrating codelivery of siRNA and paclitaxel with ultrasound-responsive nanobubbles hetero-assembled from polymeric micelles and liposomes

Drug resistance is a big problem in systemic chemotherapy of hepatocellular carcinoma (HCC), and nanomedicines loaded with both chemotherapeutic agents (e.g. paclitaxel, PTX) and siRNA's targeting antiapoptosis genes (e.g. BCL-2) possess the advantages to simultaneously overcome the efflux pump-mediated drug resistance and antiapoptosis-related drug resistance. However, tumor-penetrating drug delivery with this type of nanomedicines is extremely difficult due to their relatively big size compared to the single drug-loaded nanomedicines. Aiming at address this problem, US-responsive nanobubbles encapsulating both anti-cancer drug paclitaxel (PTX) and siRNA (PTX–NBs/siRNA) for HCC treatment were developed by hetero-assembly of polymeric micelles and liposomes in the present study. Utilizing an external low-frequency US force imposed to the tumor site, effective tumor-penetrating codelivery of siRNA and PTX was achieved via tail vein injection of PTX–NBs/siRNA into nude mice bearing human HepG2 xerografts. Consequently, the PTX treatment-inducible antiapoptosis in HepG2 cells was effectively suppressed by the codelivered siRNA targeting an antiapoptosis gene (BCL-2 siRNA) during chemotherapy. Owing to the synergistic anti-cancer effect of two therapeutic agents, tumor growth was completely inhibited using low-dose PTX in animal study. Our results highlight the great potential of this type of US-responsive hetero-assemblies carrying both anti-cancer drug and siRNA as an effective nanomedicinal system for HCC therapy.     

纳米微泡搭载siRNA联合超声靶向破碎技术干扰胶质瘤细胞增殖的体外实验研究

目的以纳米微泡超声造影剂作为基因治疗中小干扰RNA（siRNA）的载体,探讨纳米微泡搭载siRNA联合超声靶向破碎技术对siRNA的转染及干扰效率以及对抑制胶质瘤肿瘤细胞生长、提高肿瘤细胞凋亡水平的作用。 方法薄膜水化法制备粒径均一的纳米微泡,通过生物素-亲和素系统连接纳米微泡和siRNA,对胶质瘤细胞进行转染。将细胞分为纳米微泡-羧基荧光素（FAM）-乱序阴性对照siRNA（scrambled siRNA,SCR）-超声辐照组（NB-FAM-SCR-US）,纳米微泡-FAM-SCR-无超声辐照组（NB-FAM-SCR）和正常细胞组,利用激光共聚焦显微镜检测各组细胞siRNA的转染效率;将细胞分为纳米微泡-siRNA-超声辐照组（NB-siRNA-US）,纳米微泡-siRNA-无超声辐照组（NB-siRNA）,纳米微泡-SCR-超声辐照组（NB-SCR-US）,实时荧光定量PCR（Real-time PCR）及蛋白质免疫印迹（Western blot）检测各组细胞siRNA对目的基因的干扰效率;CCK-8及Annexin-V检测各组肿瘤细胞的活性及细胞凋亡率。 结果通过薄膜水化法制备的纳米微泡平均粒径为（663.9±102.5）nm,纳米微泡-siRNA的平均粒径为（707.0±127.6）nm。激光共聚焦显微镜检测发现,与NB-FAM-SCR组相比,NB-FAM-SCR-US组可在细胞核（蓝色荧光）周边观测到更多的绿色荧光,正常细胞组仅可观测到细胞核的蓝色荧光,未见绿色荧光。Real-time PCR检测发现,与NB-SCR-US组相比,NB-siRNA-US组和NB-siRNA组IDH1基因的表达在mRNA水平均降低,差异具有统计学意义（t=-20.35、-4.27,P均<0.01）;而NB-siRNA-US组目的基因表达降低更为显著,与NB-siRNA组相比,差异具有统计学意义（t=-12.34,P<0.01）。Western blot检测发现,与NB-SCR-US组和NB-siRNA组相比,NB-siRNA-US组的IDH1基因表达水平明显减低。CCK-8及Annexin-V检测各组细胞活性及细胞凋亡率,结果显示与NB-siRNA组和NB-SCR-US组相比,NB-siRNA-US组的细胞活性明显减低,细胞凋亡率明显增高,差异有统计学意义（CCK-8组：t=-13.52,-31.55,P<0.01;Annexin-V组：t=8.30、9.79,P<0.01）。 结论纳米微泡搭载siRNA联合超声靶向破碎技术,能有效提高siRNA的转染及干扰效率,可抑制肿瘤细胞生长,为胶质瘤的非侵入性治疗提供了新思路。     

Gene and oligonucleotide delivery via micro- and nanobubbles by ultrasound exposure

In recent years, the use of stimuli-responsive carriers and physical energies, such as ultrasound, magnetic force, electric force, and light, in combination therapy has attracted attention as useful gene and oligonucleotide delivery systems. These systems allow target-specific delivery to be achieved relatively easily at the application site of physical energy. Ultrasound-mediated delivery has attracted particular interest because of its noninvasive nature. Microbubbles are ultrasound contrast agents that can act as echo enhancers. Under appropriate conditions, microbubbles or nanosized bubbles can also enhance the efficiency of drug, gene, and oligonucleotide delivery by ultrasound exposure. Therefore, the combination of ultrasound technology and bubbles is expected to be a fusion diagnostic and therapeutic system known as the theranostic system. In this review, we summarize the use of micro- and nanobubbles in ultrasound-mediated gene and oligonucleotide delivery systems, and discuss their potential as therapeutic tools.