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.     

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.     

Microbubbles in ultrasound-triggered drug and gene delivery

Ultrasound contrast agents, in the form of gas-filled microbubbles, are becoming popular in perfusion monitoring; they are employed as molecular imaging agents. Microbubbles are manufactured from biocompatible materials, they can be injected intravenously, and some are approved for clinical use. Microbubbles can be destroyed by ultrasound irradiation. This destruction phenomenon can be applied to targeted drug delivery and enhancement of drug action. The ultrasonic field can be focused at the target tissues and organs; thus, selectivity of the treatment can be improved, reducing undesirable side effects. Microbubbles enhance ultrasound energy deposition in the tissues and serve as cavitation nuclei, increasing intracellular drug delivery. DNA delivery and successful tissue transfection are observed in the areas of the body where ultrasound is applied after intravascular administration of microbubbles and plasmid DNA. Accelerated blood clot dissolution in the areas of insonation by cooperative action of thrombolytic agents and microbubbles is demonstrated in several clinical trials.     

Targeting delivery of oligonucleotide and plasmid DNA to hepatocyte via galactosylated chitosan vector

Delivery of oligonucleotide to specific cells and maintenance of its biological function are important for nucleic acid therapy. The objective of this paper is to demonstrate that galactosylated low molecular weight chitosan (gal-LMWC) is a safe and effective vector of antisense oligonucleotide (ASO) and plasmid DNA for the hepatocyte targeting delivery. Gal-LMWC has been successfully prepared and MTT cytotoxic assay shows that cytotoxicity of gal-LMWC is lower than that of high molecular weight chitosan (HMWC) and low molecular weight chitosan (LMWC) in HepG2 cells. Using a complex coacervation process, gal-LMWC can form stable nano-complexes with plasmid DNA or with ASO by the electrostatic interaction. The morphometrics, particle size, and the zeta potential of gal-LMWC/ASO complexes and gal-LMWC/plasmid DNA complexes are very similar. The transfection efficiency by using gal-LMWC vector is significantly higher than that of naked DNA or naked ASO in HepG2 cells. Transfection efficiency of gal-LMWC/ASO complexes and gal-LMWC/plasmid DNA complexes depends on the molar ratio of the positive chitosan amino group and the negative DNA phosphate group (N/P ratio) strongly. Inhibition experiments confirm that the enhanced transfection efficiency is due to the ASGR mediated endocytosis of the gal-LMWC/ASO complexes or gal-LMWC/DNA complexes. These results suggest that gal-LMWC can be used in gene therapy to improve the transfection efficiency in vitro and in vivo.     

Advances in the synthesis of amphiphilic block copolymers via RAFT polymerization: stimuli-responsive drug and gene delivery

Controlled/'living' radical polymerization methods, including the versatile reversible addition-fragmentation chain transfer (RAFT) polymerization process, are rapidly moving to the forefront in construction of drug and gene delivery vehicles. The RAFT technique allows an unprecedented latitude in the synthesis of water soluble or amphiphilic architectures with precise dimensions and appropriate functionality for attachment and targeted delivery of diagnostic and therapeutic agents. This review focuses on the chemistry of the RAFT process and its potential for preparing well-defined block copolymers and conjugates capable of stimuli-responsive assembly and release of bioactive agents in the physiological environment. Recent examples of block copolymers with designed structures and segmental compositions responsive to changes in pH or temperature are reviewed and hurdles facing further development of these novel systems are discussed.     

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%).     

Systemic delivery systems of angiogenic gene by novel bubble liposomes containing cationic lipid and ultrasound exposure

Recently, we developed polyethyleneglycol (PEG)-modified liposomes (Bubble liposomes; BLs) entrapping ultrasound (US) gas and reported that the combination of BL and US exposure was an effective tool for the delivery of pDNA directly into skeletal muscles of an ischemic hindlimb model with local injection. To achieve gene delivery to deeper tissues, we attempted to prepare novel Bubble liposomes which were able to be loaded with pDNA and useful for systemic injection. We prepared BLs using cationic lipid and analyzed the interaction with the BLs and pDNA using flow cytometry. The solution of pDNA-loaded BLs (p-BLs) was further injected into the tail vein of hindlimb ischemia model mice, and transdermal US exposure was applied to ischemic hindlimb. The effects of transfection on angiogenic factors were investigated by real-time PCR. Blood flow was determined using a laser Doppler blood flow meter. The interaction with BLs and pDNA increased in the presence of DOTAP and short PEG chains and resulted in increased stability of pDNA in the serum. Transfection with pDNA encoding the bFGF gene using p-BLs and US induced various angiogenic factors and improved the blood flow. The gene delivery system into the ischemic hindlimb using the combination of p-BLs and US exposure could be an effective tool for angiogenic gene therapy via systemic injection.     

Cytosolic delivery via escape from the endosome using emulsion droplets and ultrasound

Vaporizing emulsion droplets may aid in endosomal rupture as a drug delivery route to the cytosol. Upon insonation, emulsion droplets formed from perfluorocarbon liquids may vaporize with sufficient expansion to disrupt liposomal or endosomal membranes. Emulsion droplets of perfluorohexane (PFC6) or perfluoropentane (PFC5) were prepared as free droplets in calcein or as droplets encapsulated within liposomes containing calcein. Folate-stimulated endocytosis created an experimental model, wherein calcein was self-quenched until released from the vesicles. Upon release, calcein was diluted below its self-quenching concentration and its release quantified by fluorescence. In this experimental model, folated emulsions or folated eLiposomes were incubated with folate-starved HeLa cells. Samples were exposed to two seconds of 20-kHz ultrasound (US) at 1?W/cm². Fluorescence microscopy identified released intracellular calcein. Upon insonation, both free emulsion samples and eLiposome samples produced calcein release to the cytosol. Calcein fluorescence was more intense in samples containing PFC5 compared to PFC6. Insonation of samples without emulsion droplets produced no cytosolic delivery. Likewise, cells that took up emulsion droplets but were not exposed to US did not exhibit fluorescence throughout the cell. These results suggest that vaporizing emulsion droplets are internalized into the cells and can produce endosomal escape of a therapeutic payload.     

超声引导下成人与儿童肾活检术的评价

目的评价经超声引导成人与儿童肾活检术的方法和安全性。方法83例肾脏病患者超声引导下经皮穿刺活检,经右肾或左肾下极进针,方向自外下向内上,沿肾下极皮质取材。结果68例获取足够肾组织,总成功率及3针以内成功率分别为82%、53%,成人与儿童间无差异(P>0.05)。肾活检并发症儿童以疼痛为主,成人以血尿为主。结论超声引导下成人与儿童肾活检术是安全可靠的,其病理诊断对临床治疗具有明确的指导意义。     

子宫肌瘤的超声诊断与鉴别诊断

目的：探讨子宫肌瘤的声像图特征以及诊断与鉴别诊断,以提高诊断准确率。方法：对上海中山医院青浦分院采用Acuson超声诊断仪2009～2011年诊断的213例子宫肌瘤（经腹部探测）,进行对病灶的部位、大小、形态、回声特征的监测及部分患者术后的随访观察。结果：213例患者中肌壁间肌瘤128例,浆膜下肌瘤56例,黏膜下肌瘤10例,混合性肌瘤19例。经随访,手术治疗的78例,其中病理符合76例、符合率达到97.4%左右,1例误诊（病理为子宫肌腺症）、1例漏诊（病理为妊娠合并肌瘤）,误诊率为1.3%。结论：超声可动态观察子宫肌瘤的声像图特征,具有方便快捷、误诊率低、无创伤等特点,是临床首选检查,在子宫肌瘤的诊断、治疗及术后随访方面起着相当重要的作用。     

Chitosan based micro- and nanoparticles for colon-targeted delivery of vancomycin prepared by alternative processing methods

The aim of this work was to prepare chitosan (CH) based particulate formulations for colon delivery of vancomycin (VM). Chitosan microparticles (MPs) and nanoparticles (NPs) loaded with VM were prepared using different CH/tripolyphosphate (TPP) molar ratios and different technological processes. In particular, nanoparticles were prepared by ionic gelation and freeze-drying to recover these particles, or, alternatively, by spray-drying method. Microparticles were prepared using a different spray-dryer. Micro- and nanoparticles were characterized in terms of size distributions by photon correlation spectroscopy (PCS), while encapsulation and drug loading efficiencies were studied using a dialysis method. Fourier Transform Infrared Spectroscopy (FT-IR) was employed to determine the surface composition of the micro- and nanoparticles respectively, and the morphologies of the developed systems were studied by scanning electron microscopy (SEM). Water uptake as well as drug release profiles were also measured. Antibacterial activity against Staphylococcus aureus, a Gram-positive model strain, was evaluated. FT-IR results suggested an electrostatic interaction between VM and CH/TPP particles. Moreover, the particles were found to hold a positive zeta-potential, indicating the presence of CH on the particle surfaces. Particle size and encapsulation efficiency were mainly influenced by the different manufacturing processes employed. Nanoparticles obtained by spray-drying showed the best results in terms of water uptake and drug release rate. Moreover, they showed a good bactericidal activity against S. aureus.     

超声引导下人工流产及清宫术的应用体会及推广价值

目的探讨超声引导下人工流产及清官术的应用体会及推广价值。方法对380例常规人工流产不全、药物流产不全、妊娠合并子宫肌瘤或子宫畸形、疤痕子宫合并妊娠等的官腔手术采用超声引导下全程监视宫腔内操作。结果全部一次性清除干净,宫腔内显示细条状无回声或细线状回声为标准,均取得满意效果,未发现子宫穿孔或其他并发症,术后即刻观察宫腔内有无残留物较几天后观察效果要好。结论超声引导下实施官腔操作使原来复杂、盲目、困难的官腔手术变得简单、安全、可靠,全部一次清除干净,不仅手术快、出血少,而且可以杜绝术中并发症,减少术后近期并发症及再次清官术。     

超声对小儿先天性幽门狭窄及痉挛的鉴别诊断价值

目的探讨超声对先天性肥厚性幽门狭窄和幽门痉挛的鉴别诊断价值。方法对本院已确诊为幽门狭窄的17例患儿（A组）采用超声技术测量其幽门管长度以及肌层厚度,同时与17例幽门痉挛患儿（B组）及17例正常婴儿的幽门管长度以及肌层厚度进行比较。结果A组的幽门管平均长度为（17．29＋0．64）mm,幽门管环肌平均厚度为（5．31±0．16）mm;B组的幽门管平均长度为（12．94±0．67）Inln,幽门管环肌平均厚度为（3．49±0．08）mm;对照组的幽门管平均长度为（11．25±0．64）mm,幽门管环肌平均厚度为（2．23±0．13）inln。A组的幽门管平均长度及幽门管环肌平均厚度均高于B组及对照组,各组比较差异有统计学意义（P〈0．05）,而B组与对照组在幽门管平均长度及幽门管环肌平均厚度方面差异无统计学意义（P〉0．05）。结论超声检查可以准确把握婴儿的幽门管长度及幽门管环肌厚度,对幽门狭窄及痉挛的鉴别诊断有重要意义。     

动态超声跟踪检查胎儿肾盂扩张及预后

目的探讨动态超声检查胎儿肾盂扩张及预后。方法对60例20～40周孕妇B超检查胎儿肾盂扩张情况,追踪至出生后B超检查结果进行分析。结果胎儿肾盂扩张<10mm,且胎儿无其他部位异常,均为正常胎儿。胎儿肾盂扩张≥10mm,出现肾脏病理性情况的可能性增加,如合并其他部位异常,胎儿畸形概率明显增大。结论肾盂扩张程度及有无合并其他部位畸形,可作为判断胎儿发育的预后指标。     

高糖注射在甲状腺囊肿超声介入治疗中的应用

目的评价高频超声引导下利用高糖(50%)作为硬化剂治疗甲状腺囊肿的疗效。方法利用高频超声探头引导,穿刺抽吸注入高糖治疗甲状腺囊肿。结果本组随访31例(34个囊肿),随访3个月～1年,疗效为97%,其中囊肿消失25例,占73．5%,无特殊并发症发生。结论高频超声引导下利用高糖作为硬化剂治疗甲状腺囊肿安全可靠,为甲状腺囊肿病人提供了一条新的切实可行的治疗途径。     

Lidocaine carboxymethylcellulose with gelatine co-polymer hydrogel delivery by combined microneedle and ultrasound

Abstract

A study that combines microneedles (MNs) and sonophoresis pre-treatment was explored to determine their combined effects on percutaneous delivery of lidocaine from a polymeric hydrogel formulation. Varying ratios of carboxymethylcellulose and gelatine (NaCMC/gel ranges 1:1.60–1:2.66) loaded with lidocaine were prepared and characterized for zeta potential and particle size. Additionally, variations in the formulation drying techniques were explored during the formulation stage. Ex vivo permeation studies using Franz diffusion cells measured lidocaine permeation through porcine skin after pre-treatment with stainless steel MNs and 20?kHz sonophoresis for 5-and 10-min durations. A stable formulation was related to a lower gelatine mass ratio because of smaller mean particle sizes and high zeta potential. Lidocaine permeability in skin revealed some increases in permeability from combined MN and ultrasound pre-treatment studies. Furthermore, up to 4.8-fold increase in the combined application was observed compared with separate pre-treatments after 30?min. Sonophoresis pre-treatment alone showed insignificant enhancement in lidocaine permeation during the initial 2?h period. MN application increased permeability at a time of 0.5?h for up to ～17 fold with an average up to 4 fold. The time required to reach therapeutic levels of lidocaine was decreased to less than 7?min. Overall, the attempted approach promises to be a viable alternative to conventional lidocaine delivery methods involving painful injections by hypodermic needles. The mass transfer effects were fairly enhanced and the lowest amount of lidocaine in skin was 99.7% of the delivered amount at a time of 3?h for lidocaine NaCMC/GEL 1:2.66 after low-frequency sonophoresis and MN treatment.     

上肢手术中应用超声引导下锁骨上臂丛神经阻滞的分析与研究

目的探讨上肢手术中应用超声引导下锁骨上臂丛神经阻滞的效果。方法 106例行上肢手术治疗患者为研究对象,随机分为常规组与实验组,各53例。常规组患者应用常规的锁骨上臂丛神经阻滞,实验组患者应用在超声引导下锁骨上臂丛神经阻滞。对比两组神经阻滞起效时间及神经阻滞持续时间,不良反应发生情况以及麻醉效果。结果实验组神经阻滞起效时间(3.87±1.42)min短于常规组的(6.23±2.21)min,神经阻滞持续时间(363.89±39.79)min长于常规组的(243.67±28.45)min,差异有统计学意义(P<0.05)。实验组不良反应发生率3.77%低于常规组的16.98%,差异有统计学意义(P<0.05)。实验组优良率96.23%高于常规组的84.91%,差异有统计学意义(P<0.05)。结论和常规的锁骨上臂丛神经阻滞进行对比,超声引导下锁骨上臂丛神经阻滞具有麻醉快速起效、麻醉持续时间长、不良反应发生率低等优点,值得临床广泛推广。     

超声根管预备联合热牙胶垂直加压充填对侧支根管充填的效果

目的:观察超声根管预备联合热牙胶垂直加压充填对侧支根管充填的效果.方法:将离体400颗第一前磨牙,超声根管预备后随机分两组,实验组用热牙胶垂直加压法进行根管充填;对照组用冷牙胶侧方加压法进行根管充填.根管充填完成之后,用ODIS-1型口腔数字成像系统摄片,观察记录每颗牙的侧支根管充填数目及充填材料进入侧支根管的深度.结果:两种方法根管充填侧支根管数目经χ2检验,差异有统计学意义(P<0.05);充填材料进入侧支根管的深度经t检验,差异有统计学意义(P<0.05).实验组效果均优于对照组.结论:超声根管预备联合热牙胶乖直加压充填更有利于侧支根管的封闭.     

超声联合神经刺激器引导下腰丛-腘窝坐骨神经阻滞在大隐静脉曲张手术中的应用效果

目的 观察超声联合神经刺激器引导下的腰丛联合腘窝坐骨神经阻滞在单侧大隐静脉曲张手术中的麻醉效果。方法 选择2015年1月至2015年12月在安徽医科大学附属省立医院择期行单侧大隐静脉曲张手术患者60例,ASA分级Ⅰ~Ⅱ级,采用随机数字表法将其分为神经阻滞组（NB组,30例）和硬膜外麻醉组（EA组,30例）。记录两组麻醉前（T₀）、麻醉后10分钟（T₁）、30分钟（T₂）、60分钟（T₃）的收缩压、舒张压及心率;记录两组感觉阻滞起效时间、感觉阻滞维持时间、术中麻黄碱使用情况、麻醉效果及术后随访情况（恶心呕吐及尿潴留）。结果 与EA组相比,NB组T₂时的收缩压（SBP）及舒张压（DBP）高于EA组（P<0.05）;NB组感觉阻滞起效时间小于EA组（P<0.05）,阻滞维持时间大于EA组;NB组术中麻黄碱使用例数小于EA组（P<0.05）;两组在麻醉优良率方面差异无统计学意义（P>0.05）,尿潴留NB组中的发生率低于EA组,差异有统计学意义（P<0.05）,而恶心呕吐方面比较,两组发生率的差异无统计学意义（P>0.05）。结论 超声联合神经刺激器引导下腰丛-腘窝坐骨神经阻滞用于单侧大隐静脉曲张手术的麻醉效果与硬膜外麻醉相似,且具有血流动力学影响小、术后并发症少等优点。     

Smart nanoparticles assembled by endogenous molecules for siRNA delivery and cancer therapy via CD44 and EGFR dual-targeting

We developed an anticancer siRNA delivery system (named HLPR) through modular assembly of endogenous molecules. The structure of HLPR was a tightly condensed siRNA-peptide inner core in turn surrounded by the disordered lipid layer and thin HA coating from which the EGFR-targeted amino acid sequences of YHWYGYTPQNVI partially protrude outside of cell surfaces. Both HA and YHWYGYTPQNVI anchored on HLPR were responsible for targeting CD44 and EGFR overexpressed on the tumor cell surfaces, respectively. HLPR was relatively stable in the blood circulation and reached the tumor tissue in vivo through passive and active targeting. Then HLPR entered tumor cells mainly through EGFR-mediated pathway followed by the separation of HA from the remaining parts of nanocomplexes. The HA-uncoated complexes escaped the endosome through the membrane fusion function of DOPE and released cargoes (siRNA and peptide/siRNA) in the cytoplasm. HLPR significantly inhibited the growth of implanted subcutaneous liver tumors without toxicity.