Phospholipids-based microbubbles sonoporation pore size and reseal of cell membrane cultured in vitro

OBJECTIVE: To investigate phospholipids-based microbubbles induced sonoporation and cell membrane reseal in vitro under various conditions. METHODS: A breast cancer cell line SK-BR-3 was used to investigate ultrasonic sonoporation under various conditions. Atomic force microscopy (AFM) scanning techniques were employed to observe the change of membrane pores. RESULTS: Normal SK-BR-3 cells membrane pores were evenly distributed and less than 1 microm. After ultrasound exposure, membrane pores were enlarged at different degree depending on ultrasound exposure durations, filling gas species and microbubble suspension concentration. With microbubble suspension concentration being increased to 5% or ultrasound exposure reached 30 s, membrane pores in fluorocarbon (C(3)F(8) or SF(6))-filled microbubble groups exceeded 1 microm, which were significantly larger than that of air-filled microbubble group. Membrane pores were about 2-3 microm under ultrasound 60 s with 5% fluorocarbon-filled microbubble suspension. After 24 h of incubation, most of the enlarged membrane pores could reseal to normal size, which corresponded to cell viability. CONCLUSIONS: Membrane pores can be obviously enlarged by ultrasonic sonoporation of fluorocarbon-filled microbubbles, whose reseal time depended on ultrasound exposure duration and microbubble suspension concentration.     

脂质体和脂质微泡对细胞膜的声孔作用比较

目的研究两种脂质载体——脂质体和脂质微泡对细胞膜的声孔作用。方法应用体外培养的乳腺癌细胞SK-BR-3，考察脂质体和脂质微泡在低频超声条件下，对SK-BR-3细胞膜的声孔作用，应用原子力显微镜观察超声后即刻和24 h的SK-BR-3细胞表面变化。结果超声可使细胞丧失贴壁性能，形态变圆，脂质微泡的加入明显增强这种作用。原子力显微镜观察表明，对照组(正常细胞单纯超声照射)细胞膜表面孔道暂时增大，部分细胞膜表面孔道直径大于1 μm。2%和5%脂质体+超声照射组和对照组无明显差别。脂质微泡+超声照射组对细胞膜结构有明显影响，2%脂质微泡+超声照射组产生的细胞膜表面孔道直径在1～3 μm，24 h内可以恢复。5%脂质微泡+超声照射组产生的细胞膜孔道直径2～4 μm，24 h内不能完全恢复。结论2%脂质微泡可以产生较强的声孔作用，使细胞膜上出现可逆性孔道，有利于药物或基因递送到细胞内。     

Comparison of the efficiency and toxicity of sonoporation with branched polyethylenimine-mediated gene transfection in various cultured cell lines

Objective. The objective of this study is to evaluate transfection efficiency and safety for gene delivery by sonoporation in comparison with cationic polymer gene carrier branched polyethylenimine (BPEI).

Methods. The cDNA expressing VEGF¹⁶⁵ was cloned under chicken β-actin promoter. The plasmid DNA was transfected into the CHO, HEK293, and NIH3T3 cells using microbubble-based sonoporation and BPEI (25 kDa) under various conditions. Enzyme-linked immunosorbent assay (ELISA) was used to determine the expressed protein level. Cytotoxicities of transfection methods were compared by Cell Counting Kit-8.

Results. At 1 MHz intensity, transfection efficiency of sonoporation was enhanced by microbubble concentration with no detrimental effects. By contrast, BPEI exacerbated cell viability, despite its high transgene expression efficiency.

Conclusion. Sonoporation gene therapy might be the safest technique to be used in actual clinical practice.     

Optimization of ultrasound and microbubbles targeted gene delivery to cultured primary endothelial cells

Ultrasound and microbubbles targeted gene delivery (UMTGD) is a promising technique for local gene delivery. As the endothelium is a primary target for systemic UMTGD, this study aimed at establishing the optimal parameters of UMTGD to primary endothelial cells. For this, an in vitro ultrasound (US) setup was employed in which individual UMTGD parameters were systematically optimized. The criteria for the final optimized protocol were: (1) relative high reporter gene expression levels, restricted to the US exposed area and (2) induction of not more than 5% cell death. US frequency and timing of medium replacement had a strong effect on UMTGD efficiency. Furthermore, US intensity, DNA concentration and total duration of US all affected UMTGD efficiency. Optimal targeted gene delivery to primary endothelial cells can be accomplished with Sonovue® microbubbles, using 20 μg/ml plasmid DNA, a 1 MHz US exposure of Ispta 0.10 W/cm² for 30 s with immediate medium change after UMTGD. This optimized protocol resulted in both an increase in the number of transfected cells (more than three fold) and increased levels of transgene expression per cell (170%).     

超声联合一氧化氮微泡体外干预骨髓间充质干细胞安全性探讨

目的研究超声联合一氧化氮(NO)微泡体外干预大鼠骨髓间充质干细胞(BMSCs)的安全性。方法体外分离培养大鼠BMSCs,分为超声联合NO微泡组(包括1∶70,1∶50,1∶20浓度亚组)及空白对照组,体外干预后MTT法检测BMSCs增殖,PI染色流式细胞术分析细胞凋亡及细胞周期。结果与空白对照组比较,1∶20浓度组显著抑制BMSCs增殖(P<0.05);1∶50浓度组与1∶20浓度组明显诱导细胞凋亡(P<0.05)。结论超声联合NO微泡体外干预BMSCs,1∶70浓度对其是安全的。     

Comparing encapsulation efficiency and ultrasound-triggered release for protein between phospholipid-based microbubbles and liposomes

This work was to compare the encapsulation efficiency and ultrasound-triggered release for protein between microbubbles and liposomes. Bovine serum albumin (BSA) was used as a model. Final ratios between BSA and HPC in microbubbles and liposomes were 1 : 5, 1 : 7 and 1 : 10, respectively. Morphologic characteristics and contrast enhancement of loaded microbubbles and liposomes were measured. Encapsulation efficiency and ultrasound-stimulated release profile were detected. The mean size of loaded microbubbles and liposomes was 3.4 µm and 1.7 µm, respectively. Encapsulation efficiency of microbubbles had an inverse relationship with the ratio between BSA and HPC, while loaded liposomes remained nearly unchanged in the designed range of the ratio between BSA and HPC. Microbubbles resulted in significant enhancement of CnTi images. After ultrasound, more than 90% of the entrapped BSA was released from microbubbles, but less than 5% of BSA released from liposomes. Microbubbles are a promising delivery system for proteins.     

κ-硒化角叉菜胶对大鼠红细胞膜流动性与封闭度的影响(英文)

通过测定大鼠红细胞膜荧光偏振度P和N A DH-细胞色素c氧化还原酶的变化,研究kappa-硒化卡拉胶对红细胞膜流动性和封闭度的影响。结果表明,kappa-硒化卡拉胶140mg·kg~(-1)·d~(-1)×30 d可显著降低大鼠红细胞膜荧光偏振度,即可显著提高细胞膜流动性(P<0.05),ig 140及70 mg·kg~(-1)·g~(-1)×30d可显著提高大鼠红细胞膜封闭度(P<0.05)。     

通过喹诺酮抗生素与磷脂膜的相互作用预测肺泡巨噬细胞单层的体外摄取

目的;预测肺泡巨噬细胞单层的体外摄取.方法:以培养的肺泡巨噬细胞单层为体外模型,用磷脂膜色谱,脂质体/水系统评价药物与磷脂膜的相互作用,分别表示为lg k_(1AM),lg D_(L B,7.4),用正辛醇/水系统测定参考的疏水性参数(lg D_(O/B,7.4).结果:lg D_(L B,7.4)(r~2=0.93)比lg D_(O B,7.4)(r~2=0.65)具有与lg k_(1AM)更显著的相关性.lg k_(1AM)和lg D_(L B,7.4)均 比lg D_(O/B,7.4)与细胞内药物的蓄积度有更好的相关性.但对于由5个两性喹酮抗生素和奎尼丁组成的受试集合,三者均与药物进入细胞内的速度具有相近的显著的正相关性.结论:磷脂膜色谱和脂质体/水系统给出相似的亲脂性测量尺度,且均与正辛醇/水系统区别有显著性.与疏水性参数相比,膜亲和性是更有效的肺泡巨噬细胞内药物蓄积和结合的预测参数.     

超声微泡介导的基因递送系统应用进展

超声波可聚焦于体内的特定部位。含气体微泡既可以作为医学超声显像的造影剂，又可以作为药物或基因载体。超声微泡有望实现基因的靶向递送，因此成为药物递送系统研究的热门领域。本文阐述了超声微泡介导的基因递送系统在心肌、血管、骨骼肌和肿瘤组织等方面的研究进展，讨论其在未来应用中面临的问题。     

脂质包膜微泡超声造影剂基础物理和声学性质以及体外造影增强实验

目的为了研究一种具有良好声学性质和增强造影效果的稳定微泡,并使其能够运用在心血管系统疾病的诊断中,增强超声显影效果,设计本实验。方法对这种磷脂为膜材的微泡造影剂（Lipidcoatedmicrobubble,LCM）的形态、粒径及其分布、zeta电位以及声学性质做了测定。研究了该微泡在不同浓度下的散射特性和二次谐波散射性质,并分析了浓度和散射波幅度之间的关系。考察了LCM体外增强血栓显影的效果。结果该微泡的平均粒径为3.38μm,95%的微泡粒径小于5μm。声学性质表明有造影剂的情况下,在不同的实验浓度下均能接收到收发探头分置且两探头成90°方式下的散射波,二次谐波的散射强度随着微泡浓度的降低逐渐降低,并且由微泡产生的二次谐波相对生理盐水空白产生的谐波幅度的增加率和微泡浓度成线性关系。LCM能显著增强超声显像效果,使得血栓显影更加清晰。结论该自制微泡表现出了良好的物理状态、声学性质以及造影增强效果。     

Penetration of different molecule sizes upon ultrasound combined with microbubbles in a superficial tumour model

Abstract

Ultrasound combined with microbubbles (USMB) has been extensively applied to enhance drug and gene targeting delivery. However, the accumulation and distribution of particle size in the range of 5–30?nm (nano drug) to the tumour and the effects of intratumoral vascular density on permeability have been rarely reported. This study investigated Evans blue (EB) and fluorescein isothiocyanate-labelled dextran (FITC-dextran) distribution in tumour tissue upon USMB with various molecular sizes (3.7?nm and 30.6?nm). USMB increased the penetration of molecules with sizes of 5-20?nm in the whole tumour tissue, especially on the rim. For a molecule with sizes of 30.6?nm, USMB only increased penetration around the rim of the tumour with minimal improvement in the central of tumour. USMB enhanced the permeability of tumour tissue and increased tumour cells dose exposure without affecting tumour blood perfusion or microvessel density. The current study served as the foundation of parameter preference for therapeutic USMB drug delivery.     

硫酸铝在体外对大鼠胚胎组织谷胱甘肽含量和卵黄囊细胞膜流动性的影响

目的　为探讨铝的发育毒性及机理。方法孕 9.5d大鼠胚胎于体外培养系统中给予不同剂量的硫酸铝 ,培养 4 8h后 ,观察胚胎生长发育和器官形态分化状况 ;应用二硫代双硝基苯甲酸 (DTNB)直接法测定胚胎组织谷胱甘肽 (GSH)含量 ;以 1,6 二苯己三烯为荧光探剂 ,用荧光偏振技术测定卵黄囊细胞膜脂质流动性。结果　当培养液中铝浓度为1.2mg·L- 1时 ,胚胎生长发育和分化明显被抑制 ;3.0mg·L- 1时 ,畸形胚胎发生率明显升高 ,主要有神经管闭合不全 ,脑发育不良和体翻转不全 ;6 .0mg·L- 1时 ,胚胎组织GSH含量和卵黄囊细胞膜脂质流动性显著降低。上述效应均呈现出一定的剂量 效应 (反应 )关系。结论　铝有潜在的致畸性和胚胎毒性 ,胚胎组织GSH含量和卵黄囊细胞膜流动性降低可能在铝致胚胎发育毒性中起重要作用     

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?相似文献   

Protective effect of diosmetin on in vitro cell membrane damage and oxidative stress in cultured rat hepatocytes.

Primary cultures of rat hepatocytes were used to study the effects of the flavonoids diosmin and its main metabolite diosmetin on the cell membrane damage caused by erythromycin estolate (EE) and oxidative stress caused by tert-butylhydroperoxide (TBHP). The damage was evaluated by the leakage of intracellular enzymes lactate dehydrogenase, aspartate-aminotransferase and the residual cell content of a lysosomal marker acid phosphatase (AP). After treating the cells for 40 h with diosmetin EE induced less enzyme leakage. The content of AP was kept higher by diosmetin pretreatment after 6 h exposure to EE. Diosmin at the same concentrations had barely any effect. Diosmetin, but not diosmin, also protected against TBHP toxicity and this was related to lower lipid peroxidation and higher glutathione content caused by pretreatment with the flavonoid. When the cells were treated simultaneously with TBHP and diosmetin after 21 h of culture, the protection by the flavonoid was even higher. In fact the antioxidant activity of diosmetin was considerably greater than that of diosmin. After 40 h exposure to both flavonoids diosmin but not diosmetin was detectable in the cell membrane fraction, suggesting that the latter's protective effect is associated with its metabolites.     

Preparation of chitosan microspheres using membrane emulsification and its size modelling

The chitosan microspheres were prepared by a membrane emulsification method with variations of the N₂ gas pressure and the chitosan concentration. The pressure of N₂ gas was varied within the range from 0.2?×?10⁵ to 0.8?×?10⁵?Pa at chitosan concentration 1.5?wt%. In addition, the concentration of chitosan was varied between 0.5?～?2.0?wt% at 0.4?×?10⁵?Pa of N₂ gas pressure. Using this method, it is possible to prepare stable emulsions with a very narrow droplet size distribution in comparison with conventional methods. The average size of the microspheres was dependent on the N₂ gas pressure and the concentration, that is it was increased with the pressure and the concentration. The modelling of the size for the microspheres according to the concentration was carried out using Macleod's relation and Parkins & Brown equation. The former shows the relationship between density and surface tension and the latter demonstrates the correlation between the volume of the microspheres and the interfacial tension. The modelling results were in good agreement with the experimental data to predict the microspheres size with the variation of concentration.     

Effects of temporins on molecular dynamics and membrane permeabilization in lipid vesicles

Abstract: Temporins are a novel family of small (10–13 residues) cationic antimicrobial peptides recently isolated from the skin of the European red frog Rana temporaria. Although recently acquired evidence shows that temporins have the potential to kill bacteria by permeabilizing the cytoplasmic membrane, the molecular mechanisms of membrane selectivity and permeabilization are largely unknown. In this study, it was found that temporins cause the release of fluorescent markers entrapped in phosphatidylcholine liposomes in a manner that depends significantly on the size of the solute. Temporins were also shown to lack a detergent‐like effect on lipid vesicles, indicating that marker leakage caused by these peptides is not due to total membrane disruption but to perturbation of bilayer organization on a local scale. Binding of temporins to liposomes did lead to a small increase in lipid hydrocarbon chain mobility, as revealed by EPR spectroscopy of nitroxide‐labeled fatty acids incorporated in the bilayer. Reference experiments were conducted using the bee venom peptide melittin, whose properties and behavior in natural and model membrane systems are well known. Our findings for temporins are discussed in relation to the models proposed to date to account for the action of antimicrobial peptides on membranes.     

琥珀酸胆固醇酯作为脂质体膜稳定剂的研究及其在制备柴胡皂苷-D脂质体中的应用

目的研究琥珀酸胆固醇酯(CHEMS)对二棕榈酰磷脂酰胆碱(DPPC)脂质体的膜稳定作用以及作用机制；以CHEMS和DPPC为膜材制备柴胡皂苷-D(SSD)脂质体，考察其包封率和溶血性。方法差示扫描量热法(DSC)和荧光释放实验考察CHEMS的膜稳定作用，傅立叶红外(FT-IR)研究CHEMS与DPPC膜的作用机制，沉降实验研究CHEMS与SSD相互作用，溶血实验考察了以CHEMS为膜材包裹SSD脂质体的溶血性。结果CHEMS在膜稳定性上优于胆固醇(CHOL)，CHEMS与DPPC的极性端头同时有氢键和静电作用；CHEMS与SSD不会形成不溶性复合物(INCOM)，用DPPC和CHEMS为膜材制备了稳定的SSD包裹的脂质体，其溶血性大大降低。结论CHEMS对DPPC膜的稳定作用大于CHOL，并可取代CHOL作为制备胆固醇依赖性的溶血性皂苷脂质体的膜稳定剂。     

Monitoring safety of liposomes containing rifampicin on respiratory cell lines and in vitro efficacy against Mycobacterium bovis in alveolar macrophages

Rifampicin-encapsulated liposome suspensions were prepared by a chloroform-film method and converted to dry powders by freeze-drying with mannitol as a cryoprotectant. The liposome suspension had multilamellar nanovesicles with 50% rifampicin encapsulation. The liposome dry powder comprised particles with a mass median aerodynamic diameter of 3.4?μm, with 60% present as a fine particle fraction. Rifampicin-encapsulated liposomes were evidently nontoxic to respiratory associated cells, including bronchial epithelial cells, small airway epithelial and alveolar macrophages (AMs). Furthermore, the liposomes did not activate AMs to produce interleukin-1β, tumor necrosis factor-α, and nitric oxide at a level that would cascade to other inflammatory effects. The minimum inhibitory concentrations against Mycobacterium bovis was 0.2 and 0.8?μM for liposomes containing rifampicin and free rifampicin, respectively. The less negatively charged reconstituted liposome displayed the greatest activity against intracellular growth of M. bovis.     

Improving the cardio protective effect of aFGF in ischemic myocardium with ultrasound-mediated cavitation of heparin modified microbubbles: preliminary experiment

Ultrasound (US)-mediated cavitation of microbubbles has evolved into a new tool for organ-specific gene and drug delivery. This paper was to investigate the feasibility of acidic fibroblast growth factor (aFGF) intravenous delivery to the ischemic myocardium of rats by ultrasonic microbubbles modified with heparin. Heparin modified microbubbles (HMB) were prepared by the freeze-dried method. Acute myocardial infarction (AMI) model was established and the cardio protective effect of the aFGF combing with HMB (aFGF-HMB) under US-mediated cavitation technique was investigated. aFGF-HMB combined with US-mediated cavitation technique was examined by ECG. Ejection fraction (EF), fractional shortening (FS) and left ventricular diastolic diameter (LVDd) were measured to monitor the improvement of global myocardial contractile function. Myocardial tissue was stained with hematoxylin and eosine (HE) to evaluate the elaborate general morphology of the ischemic myocardium. From morphologic observation and echocardiography in rat heart, aFGF-HMB had suitable size distribution, physical stability and good acoustic resonance function. From AMI rat experiments, aFGF-HMB under US-mediated cavitation technique exerted aFGF cardio protective effect in ischemic myocardium. From histological evaluation, US-mediated cavitation of aFGF-HMB showed improvement of myocardial ischemia. With the visual imaging and US-triggered drug release advantages, US-mediated cavitation of aFGF-HMB might be developed as a novel technique for targeting delivery of aFGF into ischemic myocardium.