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.     

Targeted delivery of a novel group of site-directed transglutaminase inhibitors to the liver using liposomes: a new approach for the potential treatment of liver fibrosis

Liver fibrosis and its end-stage disease cirrhosis are a main cause of mortality and morbidity worldwide. Thus far, there is no efficient pharmaceutical intervention for the treatment of liver fibrosis. Liver fibrosis is characterized by excessive accumulation of the extracellular matrix (ECM) proteins. Transglutaminase (TG)-mediated covalent cross-linking has been implicated in the stabilization and accumulation of ECM in a number of fibrotic diseases. Thus, the use of tissue TG2 inhibitors has potential in the treatment of liver fibrosis. Recently, we introduced a novel group of site-directed irreversible specific inhibitors of TGs. Here, we describe the development of a liposome-based drug-delivery system for the site-specific delivery of these TG inhibitors into the liver. By using anionic or neutral-based DSPC liposomes, the TG inhibitor can be successfully incorporated into these liposomes and delivered specifically to the liver. Liposomes can therefore be used as a potential carrier system for site-specific delivery of the TG2 inhibitors into the liver, opening up a potential new avenue for the treatment of liver fibrosis and its end-stage disease cirrhosis.     

Therapeutic applications of lipid-coated microbubbles

Lipid-coated microbubbles represent a new class of agents with both diagnostic and therapeutic applications. Microbubbles have low density. Stabilization of microbubbles by lipid coatings creates low-density particles with unusual properties for diagnostic imaging and drug delivery. Perfluorocarbon (PFC) gases entrapped within lipid coatings make microbubbles that are sufficiently stable for circulation in the vasculature as blood pool agents. Microbubbles can be cavitated with ultrasound energy for site-specific local delivery of bioactive materials and for treatment of vascular thrombosis. The blood-brain barrier (BBB) can be reversibly opened without damaging the neurons using ultrasound applied across the intact skull to cavitate microbubbles within the cerebral microvasculature for delivery of both low and high molecular weight therapeutic compounds to the brain. The first lipid-coated PFC microbubble product is currently marketed for diagnostic ultrasound imaging. Clinical trials are currently in process for treatment of vascular thrombosis with ultrasound and lipid-coated PFC microbubbles (SonoLysis Therapy). Targeted microbubbles and acoustically active PFC nanoemulsions with specific ligands can be developed for detecting disease at the molecular level and targeted drug and gene delivery. Bioactive compounds can be incorporated into these carriers for site-specific delivery. Our aim is to cover the therapeutic applications of lipid-coated microbubbles and PFC emulsions in this review.     

肝纤维化分子机制及治疗研究进展

肝纤维化由慢性肝炎造成,若治疗不及时将发展成为肝硬化、肝癌。目前,临床上尚无理想的治疗药物。随着肝纤维化分子机制逐渐得以阐明、基因技术不断发展,使肝纤维化的治疗成为可能。目前,肝纤维化治疗主要是通过抑制肝星状细胞（HSC）活化、增殖,抑制细胞外基质（ECM）的合成,促进ECM的降解,诱导活化HSC凋亡。本文就肝纤维化的分子机制和治疗进行综述,为以后的研究提供参考。     

Ultrasonic gene and drug delivery to the cardiovascular system

Ultrasound targeted microbubble destruction has evolved as a promising tool for organ specific gene and drug delivery. This technique has initially been developed as a method in myocardial contrast echocardiography, destroying intramyocardial microbubbles to characterize refill kinetics. When loading similar microbubbles with a bioactive substance, ultrasonic destruction of microbubbles may release the transported substance in the targeted organ. Furthermore, high amplitude oscillations of microbubbles lead to increased capillary and cell membrane permeability, thus facilitating tissue and cell penetration of the released substance. While this technique has been successfully used in many organs, its application in the cardiovascular system has dominated so far. Drug delivery using microbubbles has played a minor role in the cardiovascular system. In contrast, gene transfer has been successfully achieved in many studies. Both viral and non-viral vectors were used for loading on microbubbles. This review article will give an overview on studies that have applied ultrasound targeted microbubble destruction to deliver substances in the heart and blood vessels. It will show potential therapeutic targets, especially for gene therapy, describe feasible substances that can be loaded on microbubbles, and critically discuss prospects and limitations of this technique.     

反义核酸技术在受胶原基因表达调控的肝纤维化研究中的应用

肝纤维化是慢性肝损伤的修复反应，以胶原为主的细胞外基质（ECM）在肝内大量沉积的病理过程。其形成机制较为复杂，各种细胞因子彼此相互作用，形成细胞因子网络，共同调控肝纤维化的发生、发展。抗肝纤维化治疗策略主要包括调控HSC活化增殖或促其凋亡、抑制胶原合成或促其降解、细胞因子治疗和间充质干细胞治疗等。反义核酸技术是一种发展迅速并极富应用前景的基因控制技术。它是利用DNA或RNA分子通过Watson Crick碱基配对原则与目的基因的mRNA互补结合，通过各种机制使其降解或抑制其编码蛋白的翻译，从而抑制目的基因的表达，主要包括反义寡核苷酸技术、RNA干扰技术和三股螺旋结构寡核苷酸技术。本文综述了应用反义核酸技术调控相关基因的表达来防治肝纤维化的研究现状。     

Small extracellular vesicles encapsulating lefty1 mRNA inhibit hepatic fibrosis

Liver fibrosis is the deposition of extracellular matrix(ECM) in the liver caused by persistent chronic injury, which can lead to more serious diseases such as cirrhosis or cancer. Blocking the effect of transforming growth factor β1 (TGF-β1), one of the most important cytokines in liver fibrosis, may be one of the effective ways to inhibit liver fibrosis. As a kind of natural nano-scale vesicles, small extracellular vesicles(sEvs) have displayed excellent delivery vehicle properties. Herein, we...     

Ultrasound targeted microbubble destruction for drug and gene delivery

Background: Gas-filled microbubbles have been used as ultrasound contrast agents for some decades. More recently, such microbubbles have evolved as experimental tools for organ- and tissue-specific drug and gene delivery. When sonified with ultrasound near their resonance frequency, microbubbles oscillate. With higher ultrasound energies, oscillation amplitudes increase, leading to microbubble destruction. This phenomenon can be used to deliver a substance into a target organ, if microbubbles are co-administered loaded with drugs or gene therapy vectors before i.v. injection. Objective: This review focuses on different experimental applications of microbubbles as tools for drug and gene delivery. Different organ systems and different classes of bioactive substances that have been used in previous studies will be discussed. Methods: All the available literature was reviewed to highlight the potential of this non-invasive, organ-specific delivery system. Conclusion: Ultrasound targeted microbubble destruction has been used in various organ systems and in tumours to successfully deliver drugs, proteins, gene therapy vectors and gene silencing constructs. Many proof of principle studies have demonstrated its potential as a non-invasive delivery tool. However, too few large animal studies and studies with therapeutic aims have been performed to see a clinical application of this technique in the near future. Nevertheless, there is great hope that preclinical large animal studies will confirm the successful results already obtained in small animals.     

Ultrasound targeted microbubble destruction for drug and gene delivery

Background: Gas-filled microbubbles have been used as ultrasound contrast agents for some decades. More recently, such microbubbles have evolved as experimental tools for organ- and tissue-specific drug and gene delivery. When sonified with ultrasound near their resonance frequency, microbubbles oscillate. With higher ultrasound energies, oscillation amplitudes increase, leading to microbubble destruction. This phenomenon can be used to deliver a substance into a target organ, if microbubbles are co-administered loaded with drugs or gene therapy vectors before i.v. injection. Objective: This review focuses on different experimental applications of microbubbles as tools for drug and gene delivery. Different organ systems and different classes of bioactive substances that have been used in previous studies will be discussed. Methods: All the available literature was reviewed to highlight the potential of this non-invasive, organ-specific delivery system. Conclusion: Ultrasound targeted microbubble destruction has been used in various organ systems and in tumours to successfully deliver drugs, proteins, gene therapy vectors and gene silencing constructs. Many proof of principle studies have demonstrated its potential as a non-invasive delivery tool. However, too few large animal studies and studies with therapeutic aims have been performed to see a clinical application of this technique in the near future. Nevertheless, there is great hope that preclinical large animal studies will confirm the successful results already obtained in small animals.     

Peroxisome proliferator‐activated receptor‐δ as emerging target in liver disease

Liver fibrosis is characterized by an excessive deposition of extracellular matrix (ECM) proteins that occurs in chronic liver disease of any origin, including nonalcoholic steatohepatitis (NASH), alcohol abuse, and viral hepatitis. Cirrhosis occurs with the development of regenerating nodules of hepatocytes and is a major health burden worldwide. Patients with decompensated liver cirrhosis have a poor prognosis, with liver transplantation often being necessary. The current treatment paradigm for patients with hepatic fibrosis is to treat the underlying liver disease. However, if this cannot be achieved, there are currently no effective antifibrotic treatments for patients with chronic liver diseases. With the advent of basic molecular technology providing insight into the mechanisms of the development of hepatic fibrosis, there is now an opportunity to develop therapeutic interventions for human clinical use. In this review, the function of peroxisome proliferator‐activated receptor‐δ (PPAR δ) will be summarized with a special emphasis on ligand activation as potential use in liver disease. Drug Dev Res 2009. © 2009 Wiley‐Liss, Inc.     

Matrix Metalloproteinase Gene Delivery for Liver Fibrosis

The resolution of advanced liver fibrosis has been recently recognized to be possible, if the causative stimuli are successfully removed. However, whether complete resolution from cirrhosis, the end stage of liver fibrosis, can be achieved is still questionable. Delivery of interstitial collagenases, such as matrix metalloproteinase (MMP)-1, in the liver could be an attractive strategy to treat advanced hepatic fibrosis from the view point that the imbalance between too few interstitial collagenases and too many of their inhibitors is the main obstacle to the resolution from fibrosis. Remodeling of hepatic extracellular matrix by delivered interstitial collagenases also facilitates the disappearance of activated hepatic stellate cells, the main matrix-producing cells in the liver, and promotes the proliferation of hepatocytes. This review will focus on the impact of the gene delivery of MMPs for the treatment of advanced liver fibrosis while discussing other current therapeutic strategies for liver fibrosis, and on the need for the development of a safe and effective delivery system of MMPs.     

Microbubbles as a vehicle for gene and drug delivery: current clinical implications and future perspectives

Ultrasound targeted microbubble destruction (UTMD) has evolved as a novel system for non-invasive, organ- and tissue-specific drug and gene delivery. Initially developed as ultrasound contrast agents, microbubbles (MBs) have increasingly gained attention for their ability to directly deliver different classes of bioactive substances (e.g. genes, drugs, proteins, gene silencing constructs) to various organ systems and tumors. Bioactive substances can be attached to or incorporated in the microbubble shells. Applying ultrasound at their resonance frequency, microbubbles oscillate. When using higher ultrasound energies, oscillation amplitudes increase, finally resulting in microbubble destruction. This leads to increased capillary and cell membrane permeability in the immediate vicinity of the ruptured MBs, thus facilitating tissue and cell penetration of co-administered or loaded bioactive substances. Numerous proof of principle studies have been performed, demonstrating the broad potential of UTMD as a site-specific, non-invasive therapeutic tool, delivering microbubble payload to various target tissues and organ systems or facilitating uptake of bioactive substances into tissues or cells. This review focuses on current in vivo studies and therapeutic approaches of UTMD. Promising results give hope for future clinical applications of this novel non-viral vector system. Nevertheless, several limitations remain, which will also be discussed in this review article.     

Targeted renal therapies through microbubbles and ultrasound

Microbubbles and ultrasound enhance the cellular uptake of drugs (including gene constructs) into the kidney. Microbubble induced modifications to the size selectivity of the filtration capacity of the kidney may enable drugs to enter previously inaccessible compartments of the kidney. So far, negative renal side-effects such as capillary bleeding have been reported only in rats, with no apparent damage in larger models such as pigs and rabbits.Although local delivery is accomplished by applying ultrasound only to the target area, efficient delivery using conventional microbubbles has depended on the combined injection of both drugs and microbubbles directly into the renal artery. Conjugation of antibodies to the shell of microbubbles allows for the specific accumulation of microbubbles in the target tissue after intravenous injection. This exciting approach opens new possibilities for both drug delivery and diagnostic ultrasound imaging in the kidney.     

肝巨噬细胞与肝纤维化研究进展

肝纤维化是由各种病因所致慢性肝损伤的修复反应,其持续进展可发展为肝硬化甚至肝细胞癌,最终导致肝功能衰竭。目前,肝纤维化尚无有效治疗方法。肝巨噬细胞在肝内炎症反应、纤维化的进展和消退方面发挥关键作用,已成为抗肝纤维化的重要治疗靶细胞。主要就肝巨噬细胞在肝纤维化过程中的作用进行综述,以期能够为肝纤维化治疗提供思路。     

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。     

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.     

细胞因子基因多态性与肝纤维化发展关系的研究进展

慢性肝炎导致肝纤维化进而发展成肝硬化、肝癌等疾病的发生机制复杂,目前尚未全完阐明。细胞因子在肝纤维化发展过程起到了重要的调控作用,研究发现其基因多态性与肝纤维化的发展及药物治疗效果具有密切的联系,为临床预测和诊断以及实现个体化用药提供了依据。本文选取了与肝纤维化关系密切的细胞因子基因多态性与肝纤维化发展关系进行综述,为以后的研究提供参考。     

Effective gene delivery with novel liposomal bubbles and ultrasonic destruction technology

From the viewpoint of safety, non-viral vector systems represent an attractive gene delivery system for gene therapy. However, the transfection efficiency of non-viral vectors in vivo is generally very low. Previously, it was reported that microbubbles, utilized as imaging agents for diagnostic echocardiography, could promote gene delivery into cells when combined with ultrasound exposure. We therefore developed novel liposomal bubbles (Bubble liposomes) containing the lipid nanobubbles of perfluoropropane which is used as ultrasound imaging agent. These Bubble liposomes were smaller in diameter than conventional microbubbles and induced cavitation upon exposure to ultrasound. These results suggested that cavitation of these Bubble liposomes could be an efficient approach for delivering plasmid DNA into cells. In addition, in in vivo gene delivery, the combination of Bubble liposomes and ultrasound provided more effective gene delivery than conventional lipofection methods, further suggesting that Bubble liposomes could be effective as a non-viral vector system in in vivo gene delivery. In this review, we discuss the characteristics of Bubble liposomes and their potential utility as a gene delivery tool in vitro and in vivo.     

MicroRNAs在肝纤维化进展中的作用

肝纤维化是世界范围内高发病率和高死亡率的主要疾病之一,它是各种慢性刺激性疾病如病毒性肝炎、酒精滥用、自身免疫性疾病、代谢性疾病以及胆汁淤积性肝病等疾病长期作用于肝脏引起的共同病理现象,进一步会发展为肝硬化、肝衰竭,门脉高压甚至引起死亡,而导致肝脏结构紊乱的细胞外基质(extracellular matrix,ECM)的过度累积是肝纤维化发生发展的主要因素。microRNAs是一类22-25nt的内源性非编码小RNA,大量研究表明,microRNAs的异常表达与肝纤维化的疾病进展密切相关,该文就近些年新发现的microRNAs对肝纤维化疾病中肝星状细胞(hepatic stellate cells,HSCs)的活化、增殖、凋亡以及老化的调控作用及机制进行总结并提出展望。