Preparation of targeted microbubbles: ultrasound contrast agents for molecular imaging

Targeted ultrasound contrast agents can be prepared by attaching targeting ligands to the lipid, protein or polymer shell coating of gas-filled microbubbles. These materials are stable on storage, fully biocompatible and can be administered parenterally. Detection of microbubble contrast agents by ultrasound is very efficient (single particles with picogram mass can be visualized). Covalent or noncovalent binding techniques can be used to attach targeting ligands. Ligand-carrying microbubbles adhere to the respective molecular targets in vitro and in vivo. Several biomechanical methods are available to improve targeting efficacy, such as the use of a flexible tether spacer arm between the ligand and the bubble, and the use of folds on the microbubble shell, that project out, enhancing the contact area and increasing the length of the lever arm.     

超声－微气泡介导的基因转染研究进展

能否成功高效地转染靶基因对于基因治疗的效果具有决定性的影响。微气泡是具有稳定的封装壳,直径为微米量级的小气泡,已作为超声造影剂被广泛应用。微气泡在超声脉冲的作用下可以在靶区释放其携带的基因并使之转染,同时超声脉冲产生的热效应和空化效应能提高转染率。若将微气泡与磁性纳米颗粒结合,还可以进一步提高转染率和转染精度,是一种理想、安全的基因载体。本文综述了由超声－微气泡介导的基因转染在近5年的主要研究成果。对影响转染率的主要因素如微气泡种类、超声辐照条件、基因及受体类型等方面做了详细的论述,并对微气泡介导基因转染过程中的安全性、长效性和差异性等问题进行了讨论。     

Osteogenesis imperfecta: prospects for molecular therapeutics

Osteogenesis Imperfecta (OI) is a dominant negative disorder of connective tissue. OI patients present with bone fragility and skeletal deformity within a broad phenotypic range. Defects in the COL1A1 or COL1A2 genes, coding, respectively, for the alpha1 and alpha2 chains of type I collagen, are the causative mutations. Over 150 mutations have been characterized. Both quantitative defects, such as null COL1A1 alleles, and qualitative defects, such as glycine substitutions, exon skipping, deletions, and insertions, have been described in type I collagen. Quantitative and structural mutations are associated with the milder and more severe forms of OI, respectively. A more detailed relationship between genotype and phenotype is still incompletely understood; several models have been proposed and are being tested. Transgenic and knock-out murine models for OI have previously been created. We have recently generated a knock-in murine model (the Brittle mouse) carrying a typical glycine substitution in type I collagen. This mouse will permit a better understanding of OI pathophysiology and phenotypic variability. It will also be used for gene therapeutic approaches to OI, especially mutation suppression by hammerhead ribozymes. The present review will provide an update of OI clinical and molecular data and outline gene therapeutic approaches being tested on OI murine models for this disorder.     

Optical molecular imaging of lymph nodes using a targeted vascular contrast agent

We develop a highly specific antibody-dye conjugate for optical imaging of peripheral lymph nodes. The contrast agent consists of the monoclonal antibody recognizing endothelial ligands for the lymphocyte homing receptor L-selectin, MECA-79, and a near-infrared (near-IR) fluorescent indotricarbocyanine dye. The targeting and biodistribution behavior of MECA-79 is studied after radio-iodination and intravenous injection into mice demonstrating specific uptake in lymph nodes and accumulation in high endothelial venules (HEV). After conjugation of MECA-79 with indotricarbocyanine dye, the fluorescence imaging properties of the MECA-79 dye conjugate are examined by intravenous injection in nude mice and laser-induced fluorescence whole-body imaging in vivo. The MECA-79 antibody-dye conjugate accumulates in peripheral lymph nodes, whereas an isotype antibody-dye conjugate does not. Specific lymph node near-IR fluorescent signals become detectable within minutes after injection, and stable imaging persists for more than 24 h. The results demonstrate that vascular targeting of endothelial expression of glyocproteins is feasible to visualize the accumulation of near-IR fluorescent MECA-79 in lymph nodes, making this technology potentially useful to characterize processes of inflammation.     

Unbinding of targeted ultrasound contrast agent microbubbles by secondary acoustic forces

Targeted molecular imaging with ultrasound contrast agent microbubbles is achieved by incorporating targeting ligands on the bubble coating and allows for specific imaging of tissues affected by diseases. Improved understanding of the interplay between the acoustic forces acting on the bubbles during insonation with ultrasound and other forces (e.g. shear due to blood flow, binding of targeting ligands to receptors on cell membranes) can help improve the efficacy of this technique. This work focuses on the effects of the secondary acoustic radiation force, which causes bubbles to attract each other and may affect the adhesion of targeted bubbles. First, we examine the translational dynamics of ultrasound contrast agent microbubbles in contact with (but not adherent to) a semi-rigid membrane due to the secondary acoustic radiation force. An equation of motion that effectively accounts for the proximity of the membrane is developed, and the predictions of the model are compared with experimental data extracted from optical recordings at 15 million frames per second. A time-averaged model is also proposed and validated. In the second part of the paper, initial results on the translation due to the secondary acoustic radiation force of targeted, adherent bubbles are presented. Adherent bubbles are also found to move due to secondary acoustic radiation force, and a restoring force is observed that brings them back to their initial positions. For increasing magnitude of the secondary acoustic radiation force, a threshold is reached above which the adhesion of targeted microbubbles is disrupted. This points to the fact that secondary acoustic radiation forces can cause adherent bubbles to detach and alter the spatial distribution of targeted contrast agents bound to tissues during activation with ultrasound. While the details of the rupture of intermolecular bonds remain elusive, this work motivates the use of the secondary acoustic radiation force to measure the strength of adhesion of targeted microbubbles.     

Pathogen recognition and Toll-like receptor targeted therapeutics in innate immune cells

The innate immune system deploys a variety of pattern-recognition receptors (PRRs) which include Toll-like receptors (TLRs), RIG-I-like receptors, NOD-like receptors, and C-type lectin receptors to detect the invasion of pathogens and initiate protective responses. The intercellular and intracellular orchestration of signals from different PRRs, their endogenous or microbial ligands and accessory molecules determine the stimulatory or inhibitory responses. Progressing over the last two decades, considerable research on the molecular mechanisms underlying host–pathogen interactions has led to a paradigm shift of our understanding of TLR signaling in the innate immune system. Given that a significant amount of evidence implicates TLRs in the pathogenesis of immune diseases and cancer, and their activation occurs early in the inflammatory cascade, they are attractive targets for novel therapeutic agents. In this review, we discuss the recent advances in TLR signaling cross talks and the mechanism of pathogen recognition with special emphasis on the role of TLRs in tumor immunity and TLR-targeted therapeutics.     

Mitochondrial oxidative damage in aging and Alzheimer's disease: implications for mitochondrially targeted antioxidant therapeutics

The overall aim of this article is to review current therapeutic strategies for treating AD, with a focus on mitochondrially targeted antioxidant treatments. Recent advances in molecular, cellular, and animal model studies of AD have revealed that amyloid precursor protein derivatives, including amyloid beta (A beta) monomers and oligomers, are likely key factors in tau hyperphosphorylation, mitochondrial oxidative damage, inflammatory changes, and synaptic failure in the brain tissue of AD patients. Several therapeutic strategies have been developed to treat AD, including anti-inflammatory, antioxidant, and antiamyloid approaches. Among these, mitochondrial antioxidant therapy has been found to be the most efficacious in reducing pathological changes and in not producing adverse effects; thus, mitochondrial antioxidant therapy is promising as a treatment for AD patients. However, a major limitation in applying mitochondrial antioxidants to AD treatment has been the inability of researchers to enhance antioxidant levels in mitochondria. Recently, however, there has been a breakthrough. Researchers have recently been able to promote the entry of certain antioxidants-including MitoQ, MitoVitE, MitoPBN, MitoPeroxidase, and amino acid and peptide-based SS tetrapeptides-into mitochondria, several hundred-fold more than do natural antioxidants. Once in the mitochondria, they rapidly neutralize free radicals and decrease mitochondrial toxicity. Thus, mitochondrially targeted antioxidants are promising candidates for treating AD patients.     

Angiogenesis in gliomas: imaging and experimental therapeutics

Much of the interest in angiogenesis and hypoxia has led to investigating diagnostic imaging methodologies and developing efficacious agents against angiogenesis in gliomas. In many ways, because of the cytostatic effects of these agents on tumor growth and tumor-associated endothelial cells, the effects of therapy are not immediately evident. Hence finding clinically applicable imaging tools and pathologic surrogate markers is an important step in translating glioma biology to therapeutics. There are a variety of strategies in the approach to experimental therapeutics that target the hypoxia-inducible factor pathway, the endogenous antiangiogenic and proangiogenic factors and their receptors, adhesion molecules, matrix proteases and cytokines, and the existing vasculature. We discuss the rationale for antiangiogenesis as a treatment strategy, the preclinical and clinical assessment of antiangiogenic interventions and finally focus on the various treatment strategies, including combining antiangiogenic drugs with radiation and chemotherapy.     

超声换能器在医学成像中的发展

换能器是超声波发射和回波接收器件,是超声医学成像中最重要的声学部件.在医疗超声设备中,换能器直接影响了超声医学成像质量.概述了各种超声医学成像换能器现状,以及换能器聚焦和数字波束成像技术.     

超声换能器在医学成像中的发展

换能器是超声波发射和回波接收器件,是超声医学成像中最重要的声学部件。在医疗超声设备中,换能器直接影响了超声医学成像质量。概述了各种超声医学成像换能器现状,以及换能器聚焦和数字波束成像技术。     

微泡型超声造影剂制备及其靶向应用研究进展

超声诊断技术是世界上最广泛的影像技术之一，随着生物医学和高分子学科的发展，超声造影剂无论从制备方法、材料还是应用范围都发生了很大的变化。从制备方法、材料及其靶向应用等多方面对超声造影剂作简单的介绍。     

Amphiphilic protein micelles for targeted in vivo imaging

A variety of polymeric nanoparticles have been developed for bioimaging applications. This study reports on the use of a 50 nm recombinant protein nanoparticle with a multivalent surface as a vehicle for functionalization with a model imaging agent. Multiple fluorescent probes were covalently conjugated to surface amines of crosslinked amphiphilic elastin-mimetic protein micelles using N-hydroxysuccinimide ester chemistry. In vivo fluorescence imaging confirmed that protein micelles selectively accumulated at sites of angioplasty induced vessel wall injury, presumably via an enhanced permeability and retention effect. This investigation demonstrates the potential of amphiphilic protein micelles to be used as a vehicle for selective imaging of sites associated with a disrupted or leaky endothelium.     

Advances in preclinical therapeutics development using small animal imaging and molecular analyses: the gastrointestinal stromal tumors model

The large use of target therapies in the treatment of gastrointestinal stromal tumors (GISTs) highlighted the urgency to integrate new molecular imaging technologies, to develop new criteria for tumor response evaluation and to reach a more comprehensive definition of the molecular target. These aspects, which come from clinical experiences, are not considered enough in preclinical research studies which aim to evaluate the efficacy of new drugs or new combination of drugs with molecular target. We developed a xenograft animal model GIST882 using nude mice. We evaluated both the molecular and functional characterization of the tumor mass. The mutational analysis of KIT receptor of the GIST882 cell lines and tumor mass showed a mutation on exon 13 that was still present after in vivo cell growth. The glucose metabolism and cell proliferation was evaluated with a small animal PET using both FDG and FLT. The experimental development of new therapies for GIST treatment requires sophisticated animal models in order to represent the tumor molecular heterogeneity already demonstrated in the clinical setting and in order to evaluate the efficacy of the treatment also considering the inhibition of tumor metabolism, and not only considering the change in size of tumors. This approach of cancer research on GISTs is crucial and essential for innovative perspectives that could cross over to other types of cancer.     

DNA repair proteins as molecular therapeutics for oxidative and alkylating lung injury

Endogenous and environmental oxidation is increasingly becoming an important factor associated with numerous disorders in both children and adults. The lung is particularly prone to oxidation, as the gas exchange organ is continuously exposed to a great deal of airborne oxidants. Lung oxidation-induced toxicity is a critical clinical problem that is currently lacking cure. For example, treatment for acute respiratory distress syndrome (ARDS), a common type of acute diffuse lung injury, is strictly supportive. Alkylating chemotherapeutics and many methyl chemicals can cause acute or chronic lung injury, which is also difficult to treat. Many new approaches are being tried to improve the treatment of lung oxidation and alkylation; one of these is the use of DNA repair proteins, such as base excision repair proteins that are largely involved in repairing DNA damage caused by oxidation and alkylation. Recent advances have revealed their promising potential for treating oxidation toxicity. Here we discuss discoveries that have led to this possibility, including pioneering research into the cellular signaling transduction and molecular mechanisms of DNA repair proteins. In conclusion, when combined with other therapeutic measures such as anti-oxidant chemicals and enzymes, DNA repair proteins may have great potential for treating acute and chronic lung toxicity induced by oxidation and alkylation.     

Genetically targeted fluorogenic macromolecules for subcellular imaging and cellular perturbation

The alteration of cellular functions by anchoring macromolecules to specified organelles may reveal a new area of therapeutic potential and clinical treatment. In this work, a unique phenotype was evoked by influencing cellular behavior through the modification of subcellular structures with genetically targetable macromolecules. These fluorogen-functionalized polymers, prepared via controlled radical polymerization, were capable of exclusively decorating actin, cytoplasmic, or nuclear compartments of living cells expressing localized fluorgen-activating proteins. The macromolecular fluorogens were optimized by establishing critical polymer architecture-biophysical property relationships which impacted binding rates, binding affinities, and the level of internalization. Specific labeling of subcellular structures was realized at nanomolar concentrations of polymer, in the absence of membrane permeabilization or transduction domains, and fluorogen-modified polymers were found to bind to protein intact after delivery to the cytosol. Cellular motility was found to be dependent on binding of macromolecular fluorogens to actin structures causing rapid cellular ruffling without migration.     

Computers in ultrasonic imaging

This article describes the role of computers and digital electronics in state-of-the-art diagnostic ultrasound scanners. An overview of the computational requirements is provided, and limits on color flow image frame rates are discussed. The new scanner architectures emerging may be used to extend current limitations of ultrasonography, making features such as automatic phase aberration correction, speckle reduction, and tissue characterization available.     

In vivo molecular targeted radiotherapy

Unsealed radionuclides have been in clinical therapeutic use for well over half a century. Following the early inappropriate clinical administrations of radium salts in the early 20th century, the first real clinical benefits became evident with the use of (131)I-sodium iodide for the treatment of hypothyroidism and differentiated thyroid carcinoma and (32)P-sodium phosphate for the treatment of polycythaemia vera. In recent years the use of bone seeking agents (89)Sr, (153)Sm and (186)Re for the palliation of bone pain have become widespread and considerable progress has been evident with the use of (131)I-MIBG and (90)Y-somatostatin receptor binding agents. Although the use of monoclonal antibody based therapeutic products has been slow to evolve, the start of the 21st century has witnessed the first licensed therapeutic antibody conjugates based on (90)Y and (131)I for the treatment of non-Hodgkin's lymphoma. The future clinical utility of this form of therapy will depend upon the development of radiopharmaceutical conjugates capable of selective binding to molecular targets. The availability of some therapeutic radionuclides such as (188)Re produced from the tungsten generator system which can produce activity as required over many months, may make this type of therapy more widely available in some remote and developing countries.Future products will involve cytotoxic radionuclides with appropriate potency, but with physical characteristics that will enable the administration of therapeutic doses with the minimal need for patient isolation. Further developments are likely to involve molecular constructs such as aptamers arising from new developments in biotechnology.Patient trials are still underway and are now examining new methods of administration, dose fractionation and the clinical introduction of alpha emitting radiopharmaceutical conjugates. This review outlines the history, development and future potential of these forms of therapy.