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.     

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.     

Response of contrast agents to ultrasound

Microbubbles are used as ultrasonic contrast agents that enhance the ultrasound signals of the vascular bed. The recent development of site-targeted microbubbles opened up the possibility for molecular imaging as well as localised drug and gene delivery. Initially the microbubbles' physical properties and their response to the ultrasound beam were not fully understood. However, the introduction of fast acquisition microscopy has allowed the observation of the microbubble behaviour in the presence of ultrasound. In addition, acoustical techniques can determine the scatter of single microbubbles. Sonoporation experiments promise high-specificity drug and gene delivery, but the responsible physical mechanisms, particularly for in vivo applications, are not fully understood. An improvement of microbubble technology may address variability related problems in both imaging and drug/gene delivery.     

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.     

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.     

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。     

Nanoparticle targeting to diseased vasculature for imaging and therapy

Significant challenges remain in targeting drugs to diseased vasculature; most important being rapid blood flow with high shear, limited availability of stable targets, and heterogeneity and recycling of cellular markers. We developed nanoparticles (NPs) to target degraded elastic lamina, a consistent pathological feature in vascular diseases. In-vitro organ and cell culture experiments demonstrated that these NPs were not taken up by cells, but instead retained within the extracellular space; NP binding was proportional to the extent of elastic lamina damage. With three well-established rodent models of vascular diseases such as aortic aneurysm (calcium chloride mediated aortic injury in rats), atherosclerosis (fat-fed apoE-/- mice), and vascular calcification (warfarin + vitamin K injections in rats), we show precise NPs spatial targeting to degraded vascular elastic lamina while sparing healthy vasculature when NPs were delivered systemically. Nanoparticle targeting degraded elastic lamina is attractive to deliver therapeutic or imaging agents to the diseased vasculature.From the Clinical EditorThis novel work focuses on nanoparticle targeting of degraded elastic lamina in a variety of diseases, including atherosclerosis, vascular calcification, and aneurysm formation, and demonstrates the feasibility to deliver therapeutic or imaging agents to the diseased vasculature.     

Intratumoral evaluation of 3D microvasculature and nanoparticle distribution using a gadolinium-dendron modified nano-liposomal contrast agent with magnetic resonance micro-imaging

The enhanced permeability and retention (EPR) effect is variable depending on nanoparticle properties and tumor/vessel conditions. Thus, intratumoral evaluations of the vasculature and nanoparticle distribution are important for predicting the therapeutic efficacy and the intractability of tumors. We aimed to develop a tumor vasculature evaluation method and high-resolution nanoparticle delivery imaging using magnetic resonance (MR) micro-imaging technology with a gadolinium (Gd)-dendron assembled liposomal contrast agent. Using the Gd-liposome and a cryogenic receiving coil, we achieved 50-μm isotropic MR angiography with clear visualization of tumor micro-vessel structure. The Gd-liposome-enhanced MR micro-imaging revealed differences in the vascular structures between Colon26- and SU-DHL6-grafted mice models. The vessel volumes and diameters measured for both tumors were significantly correlated with histological observations. The MR micro-imaging methods facilitate the evaluation of intratumoral vascularization patterns, the quantitative assessment of vascular-properties that alter tumor malignancy, particle retentivity, and the effects of treatment.     

Ultrasound-mediated gene delivery

Importance of the field: The use of ultrasound with microbubbles raises the possibility of an efficient and safe gene delivery.

Areas covered in this review: This review summarizes the current state of the art of gene delivery by sonoporation under the following topics. First, the basic ultrasound parameters and the characteristics of microbubble in biological systems are discussed. Second, the extensions of sonoporation to other fields of gene delivery such as viral and non-viral vector are briefly reviewed. Finally, recent applications in an animal model for various diseases are introduced.

What the reader will gain: Information and comments on gene delivery by sonoporation or enhanced cell membrane permeability by means of ultrasound.

Take home message: Ultrasound-mediated gene delivery combined with microbubble agents provides significant safety advantages over other methods of local gene delivery.     

Advances in ultrasound-targeted microbubble-mediated gene therapy for liver fibrosis

Hepatic fibrosis develops as a wound-healing scar in response to acute and chronic liver inflammation and can lead to cirrhosis in patients with chronic hepatitis B and C. The condition arises due to increased synthesis and reduced degradation of extracellular matrix (ECM) and is a common pathological sequela of chronic liver disease. Excessive deposition of ECM in the liver causes liver dysfunction, ascites, and eventually upper gastrointestinal bleeding as well as a series of complications. However, fibrosis can be reversed before developing into cirrhosis and has thus been the subject of extensive researches particularly at the gene level. Currently, therapeutic genes are imported into the damaged liver to delay or prevent the development of liver fibrosis by regulating the expression of exogenous genes. One technique of gene delivery uses ultrasound targeting of microbubbles combined with therapeutic genes where the time and intensity of the ultrasound can control the release process. Ultrasound irradiation of microbubbles in the vicinity of cells changes the permeability of the cell membrane by its cavitation effect and enhances gene transfection. In this paper, recent progress in the field is reviewed with emphasis on the following aspects: the types of ultrasound microbubbles, the construction of an ultrasound-mediated gene delivery system, the mechanism of ultrasound microbubble–mediated gene transfer and the application of ultrasound microbubbles in the treatment of liver fibrosis.     

Gene recombinant bone marrow mesenchymal stem cells as a tumor-targeted suicide gene delivery vehicle in pulmonary metastasis therapy using non-viral transfection

One of the main limitations of anti-tumor gene therapy is the lack of an effective way to deliver therapeutic genes to tumor sites. Bone marrow mesenchymal stem cells (BMSCs) have been proposed as cellular delivery vehicles to tumor sites in tumor-targeted cancer gene therapy. Here, we investigated the therapeutic effects of cytomegalovirus-thymidine kinase expressing BMSCs (TK-BMSCs) on pulmonary melanoma metastasis combined with prodrug ganciclovir. BMSCs were successfully engineered through a non-viral gene vector. The gene recombinant BMSCs migrated to the pulmonary area and were found to have the tendency to target tumor nodules after systemic delivery. In vitro results demonstrate that the engineered BMSCs have significant suicide effects in the presence of ganciclovir in a dose-dependent manner and can exert a sufficient bystander effect on B16F10 tumor cells in co-culture experiments. In vivo studies confirmed the therapeutic effects of TK-BMSCs/ganciclovir on the metastasis tumor model.From the Clinical EditorThis study investigates the possibility of gene transfer via bone marrow mesenchymal stem cells in anti-cancer gene therapy using a metastatic melanoma model and cytomegalovirus-thymidine kinase expressing stem cells, demonstrating clear therapeutic effects.     

Targeted Endothelial Gene Delivery by Ultrasonic Destruction of Magnetic Microbubbles Carrying Lentiviral Vectors

Purpose  

Site specific vascular gene delivery is a promising tool for treatment of cardiovascular diseases. By combining ultrasound mediated microbubble destruction with site specific magnetic targeting of lentiviruses, we aimed to develop a technique suitable for systemic application.     

ICS-283: a system for targeted intravenous delivery of siRNA

ICS-283 was developed within Intradigm Corporation as a system that is designed for the systemic delivery of therapeutic small interfering (siRNA) to sites of pathological angiogenesis. The non-viral siRNA delivery system is based on synthetic nanoparticles, known as Targe (Intradigm Corporation), which functions as a broad-platform technology to deliver siRNA to specific target cells in diseased tissues. The system is constructed to incorporate different functionalities that address critical needs for successful nucleic acid delivery. The TargeTran synthetic vector is a self-assembling, layered nanoparticle that protects and targets siRNA to specific cell types in pathological tissues. At present, ICS-283 is the only antiangiogenic siRNA delivery system that is designed for intravenous administration to treat angiogenesis-driven diseases.     

Microbubble drug conjugate and focused ultrasound blood brain barrier delivery of AAV-2 SIRT-3

BackgroundDelivery of viral vectors as gene therapies to treat neurodegenerative diseases has been hampered by the inability to penetrate the blood brain barrier (BBB) and invasive or non-targeted delivery options prone to inducing immune responses. MR guided focused ultrasound (MR-g-FUS) and microbubbles have demonstrated safe, temporary, targeted BBB permeabilization clinically.MethodsWe developed clinically scalable, microbubble drug conjugates (MDCs) for the viral gene therapy, AAV.SIRT3-myc [adeno-associated virus expressing myc-tagged SIRT3], which has previously been shown to have disease modifying effects in animal models of Parkinson’s disease (PD). The lipid shells of the perfluorocarbon gas MDCs were covalently conjugated to antibodies with binding specificity to AAVs. Following systemic (iv) delivery of AAV.SIRT3-myc MDCs, MR-g-FUS was used to deliver SIRT3-myc to brain regions affected in PD. SIRT3-myc expression was determined post mortem, using immunohistochemistry.ResultsAn in vitro, SH-SY5Y cell culture model was used to show that the localized destruction of MDCs using ultrasound exposures within biological safety limits dissociated AAV2-GFP (green fluorescent protein) from the MDCs in the targeted area while maintaining their transduction capacity. In rats, MR-g-FUS resulted in BBB permeabilization in the striatum and substantia nigra (SNc). SIRT3-myc was expressed in the striatum, but not the SNc.ConclusionThese studies demonstrate that MDCs combined with MR-g-FUS are an effective method for delivery of viral vector gene therapies, such as AAV.SIRT3, to brain regions affected in PD. This technology may prove useful as a disease-modifying strategy in PD and other neurodegenerative disorders.     

ICS-283: a system for targeted intravenous delivery of siRNA

ICS-283 was developed within Intradigm Corporation as a system that is designed for the systemic delivery of therapeutic small interfering (siRNA) to sites of pathological angiogenesis. The non-viral siRNA delivery system is based on synthetic nanoparticles, known as TargeTran? (Intradigm Corporation), which functions as a broad-platform technology to deliver siRNA to specific target cells in diseased tissues. The system is constructed to incorporate different functionalities that address critical needs for successful nucleic acid delivery. The TargeTran synthetic vector is a self-assembling, layered nanoparticle that protects and targets siRNA to specific cell types in pathological tissues. At present, ICS-283 is the only antiangiogenic siRNA delivery system that is designed for intravenous administration to treat angiogenesis-driven diseases.     

Ultrasonic drug delivery--a general review

Ultrasound has an ever-increasing role in the delivery of therapeutic agents, including genetic material, protein and chemotherapeutic agents. Cavitating gas bodies, such as microbubbles, are the mediators through which the energy of relatively non-interactive pressure waves is concentrated to produce forces that permeabilise cell membranes and disrupt the vesicles that carry drugs. Thus, the presence of microbubbles enormously enhances ultrasonic delivery of genetic material, proteins and smaller chemical agents. Numerous reports show that the most efficient delivery of genetic material occurs in the presence of cavitating microbubbles. Attaching the DNA directly to the microbubbles, or to gas-containing liposomes, enhances gene uptake even further. Ultrasonic-enhanced gene delivery has been studied in various tissues, including cardiac, vascular, skeletal muscle, tumour and even fetal tissue. Ultrasonic-assisted delivery of proteins has found most application in transdermal transport of insulin. Cavitation events reversibly disrupt the structure of the stratus corneum to allow transport of these large molecules. Other hormones and small proteins could also be delivered transdermally. Small chemotherapeutic molecules are delivered in research settings from micelles and liposomes exposed to ultrasound. Cavitation appears to play two roles: it disrupts the structure of the carrier vesicle and releases the drug; and makes cell membranes and capillaries more permeable to drugs. There remains a need to better understand the physics of cavitation of microbubbles and the impact that such cavitation has on cells and drug-carrying vesicles.     

Nanotechnology in cancer therapy

Abstract

Cancer is one of the major causes of mortality worldwide and advanced techniques for therapy are urgently needed. The development of novel nanomaterials and nanocarriers has allowed a major drive to improve drug delivery in cancer. The major aim of most nanocarrier applications has been to protect the drug from rapid degradation after systemic delivery and allowing it to reach tumor site at therapeutic concentrations, meanwhile avoiding drug delivery to normal sites as much as possible to reduce adverse effects. These nanocarriers are formulated to deliver drugs either by passive targeting, taking advantage of leaky tumor vasculature or by active targeting using ligands that increase tumoral uptake potentially resulting in enhanced antitumor efficacy, thus achieving a net improvement in therapeutic index. The rational design of nanoparticles plays a critical role since structural and physical characteristics, such as size, charge, shape, and surface characteristics determine the biodistribution, pharmacokinetics, internalization and safety of the drugs. In this review, we focus on several novel and improved strategies in nanocarrier design for cancer therapy.     

Understanding ultrasound induced sonoporation: Definitions and underlying mechanisms

In the past two decades, research has underlined the potential of ultrasound and microbubbles to enhance drug delivery. However, there is less consensus on the biophysical and biological mechanisms leading to this enhanced delivery. Sonoporation, i.e. the formation of temporary pores in the cell membrane, as well as enhanced endocytosis is reported. Because of the variety of ultrasound settings used and corresponding microbubble behavior, a clear overview is missing. Therefore, in this review, the mechanisms contributing to sonoporation are categorized according to three ultrasound settings: i) low intensity ultrasound leading to stable cavitation of microbubbles, ii) high intensity ultrasound leading to inertial cavitation with microbubble collapse, and iii) ultrasound application in the absence of microbubbles. Using low intensity ultrasound, the endocytotic uptake of several drugs could be stimulated, while short but intense ultrasound pulses can be applied to induce pore formation and the direct cytoplasmic uptake of drugs. Ultrasound intensities may be adapted to create pore sizes correlating with drug size. Small molecules are able to diffuse passively through small pores created by low intensity ultrasound treatment. However, delivery of larger drugs such as nanoparticles and gene complexes, will require higher ultrasound intensities in order to allow direct cytoplasmic entry.     

Specific targets in tumor tissue for the delivery of therapeutic genes

Gene therapy is part of a growing field in molecular medicine, which will gain importance in the treatment of human diseases. Until now, almost two thirds of all clinical trials performed in gene therapy are directed against Cancer As solid tumors exceeding a certain size rely on blood supply, the administration of particulate gene delivery vectors via the bloodstream is a promising concept. Tumor cells and the tumor vasculature both offer specific molecular targets, which can be utilized for the site directed delivery of therapeutic genes. Passive targeting of macromolecular drugs including gene delivery vectors to tumors can be achieved by the so called enhanced permeability and retention (EPR) effect. The specificity can be markedly enhanced when tumor targeting ligands are used. Viral vectors, which usually do not have a natural tropism for tumor tissue, were generated to carry tumor targeting molecules on their surface. Synthetic gene delivery vectors, based on cationic lipids or cationic polymers were biochemically modified to incorporate ligands specific for tumor cells or tumor vasculature. For systemic application, these delivery systems have to fulfill certain conditions. The delivery vector should not induce any immunogenic and inflammatory responses. Several studies were conducted to reduce the immunogenicity of viral vectors; surface modification of non-viral gene delivery systems reduced their non-specific interaction with blood components. On the genetic level, tumor specific promoters add additional layers of specificity restricting the transgene expression to the tumor tissue. This review will cover the systemic application of particulate gene transfer vectors targeted to tumors and will give an overview of therapeutic concepts for cancer gene therapy.