Ultrasound guided site specific gene delivery system using adenoviral vectors and commercial ultrasound contrast agents

We have evaluated if ultrasound imaging (US) and various commercially available contrast microbubbles can serve as a non-invasive systemically administered delivery vehicle for site-specific adenoviral-mediated gene transfer in vitro and in vivo. The contrast agents were tested for their ability to enclose and to protect an adenoviral vector carrying the GFP marker gene (Ad-GFP) into the microbubbles. We have also evaluated the ability of the innate immune system to inactivate free adenoviruses as well as unenclosed viruses adsorbed on the surface of the contrast agents and in turn the ability of the microbubbles to enclose and to protect the viral vectors from such agents. In vitro as well as in vivo, innate components of the immune system were able to serve as inactivating agents to clear free viral particles and unenclosed adenoviruses adsorbed on the microbubbles' surface. Systemic delivery of Ad-GFP enclosed into microbubbles in the tail vein of nude mice resulted in specific targeting of the GFP transgene. Both fluorescence microscopy and GFP immunohistochemistry demonstrated US guided specific transduction in the targeted cells only, with no uptake in either heart, lungs or liver using complement-pretreated Ad-GFP microbubbles. This approach enhances target specificity of US microbubble destruction as a delivery vehicle for viral-mediated gene transfer.     

A New Brain Drug Delivery Strategy: Focused Ultrasound-Enhanced Intranasal Drug Delivery

Central nervous system (CNS) diseases are difficult to treat because of the blood-brain barrier (BBB), which prevents most drugs from entering into the brain. Intranasal (IN) administration is a promising approach for drug delivery to the brain, bypassing the BBB; however, its application has been restricted to particularly potent substances and it does not offer localized delivery to specific brain sites. Focused ultrasound (FUS) in combination with microbubbles can deliver drugs to the brain at targeted locations. The present study proposed to combine these two different platform techniques (FUS+IN) for enhancing the delivery efficiency of intranasally administered drugs at a targeted location. After IN administration of 40 kDa fluorescently-labeled dextran as the model drug, FUS targeted at one region within the caudate putamen of mouse brains was applied in the presence of systemically administered microbubbles. To compare with the conventional FUS technique, in which intravenous (IV) drug injection is employed, FUS was also applied after IV injection of the same amount of dextran in another group of mice. Dextran delivery outcomes were evaluated using fluorescence imaging of brain slices. The results showed that FUS+IN enhanced drug delivery within the targeted region compared with that achieved by IN only. Despite the fact that the IN route has limited drug absorption across the nasal mucosa, the delivery efficiency of FUS+IN was not significantly different from that of FUS+IV. As a new drug delivery platform, the FUS+IN technique is potentially useful for treating CNS diseases.     

Endothelial adhesion of targeted microbubbles in both small and great vessels using ultrasound radiation force

The effectiveness of microbubble-mediated ultrasound molecular imaging and drug delivery has been significantly affected by the axial laminar flow of vessels which prevents ultrasound contrast agents (UCAs) from targeting vascular endothelium. Studies show that acoustic manipulation could increase targeted UCA adhesion in microcirculation and some small vessels. In this study we demonstrate that ultrasound radiation force (USRF) can also significantly enhance the targeted adhesion of microbubbles in both small and great vessels. Our results indicate that the UCA adhesion targeted to ICAM-1 expressed on mouse cremaster microvascular endothelial cells increase about 9-fold when USRF is applied at 1 MHz and 73.9 kPa. The adhesion of anti-CD34 microbubbles to the endothelia of rat abdominal aorta was visually analyzed using scanning electron microscopy for the first time and thousands of microbubbles were found attached to the aortic endothelia after USRF application at the same acoustic parameters. Our data illustrate that targeted adhesion of anti-CD34 microbubbles is possible in normal abdominal aorta and we demonstrate the potential of using USRF in molecular imaging of a vascular target.     

Ultrasound enhanced delivery of molecular imaging and therapeutic agents in Alzheimer's disease mouse models

Alzheimer's disease is a neurodegenerative disorder typified by the accumulation of a small protein, beta-amyloid, which aggregates and is the primary component of amyloid plaques. Many new therapeutic and diagnostic agents for reducing amyloid plaques have limited efficacy in vivo because of poor transport across the blood-brain barrier. Here we demonstrate that low-intensity focused ultrasound with a microbubble contrast agent may be used to transiently disrupt the blood-brain barrier, allowing non-invasive, localized delivery of imaging fluorophores and immunotherapeutics directly to amyloid plaques. We administered intravenous Trypan blue, an amyloid staining red fluorophore, and anti-amyloid antibodies, concurrently with focused ultrasound therapy in plaque-bearing, transgenic mouse models of Alzheimer's disease with amyloid pathology. MRI guidance permitted selective treatment and monitoring of plaque-heavy anatomical regions, such as the hippocampus. Treated brain regions exhibited 16.5+/-5.4-fold increase in Trypan blue fluorescence and 2.7+/-1.2-fold increase in anti-amyloid antibodies that localized to amyloid plaques. Ultrasound-enhanced delivery was consistently reproduced in two different transgenic strains (APPswe:PSEN1dE9, PDAPP), across a large age range (9-26 months), with and without MR guidance, and with little or no tissue damage. Ultrasound-mediated, transient blood-brain barrier disruption allows the delivery of both therapeutic and molecular imaging agents in Alzheimer's mouse models, which should aid pre-clinical drug screening and imaging probe development. Furthermore, this technique may be used to deliver a wide variety of small and large molecules to the brain for imaging and therapy in other neurodegenerative diseases.     

Targeted ultrasound contrast agents: in vitro assessment of endothelial dysfunction and multi-targeting to ICAM-1 and sialyl Lewisx

An ultrasound-based molecular imaging technique capable of detecting endothelial cell markers of inflammation may allow early, non-invasive assessment of vascular disease. Clinical application of targeted, acoustically-active microbubbles requires optimization of microbubble-endothelial adhesion strength to maximize image signal-to-noise ratio, as well as the ability to discern the degree of inflammation along a continuum of dysfunction. Accordingly, we hypothesized that adhesion of intercellular adhesion molecule-1 (ICAM-1)-targeted microbubbles is dependent on the degree of endothelial inflammation, and that microbubbles multi-targeted to both ICAM-1 (via anti-ICAM-1 antibodies) and selectins (via sialyl Lewisx) demonstrate greater adhesion strength than microbubbles targeted to either inflammatory marker alone. In a radial flow chamber, microbubbles were perfused across endothelial cells activated with interleukin-1beta to four different levels of inflammation, as assessed by quantitative ICAM-1 expression. ICAM-1-targeted microbubble adhesion strength increased with increasing degree of inflammation, with a relationship that was both positive and linear (r > 0.99). Microbubble adhesion strength was significantly higher for the multi-targeted microbubbles than either of the single-targeted microbubbles. These data thus demonstrate that multi-targeting of contrast microbubbles may offer improved adhesion characteristics, allowing for greater sensitivity to inflammation. Furthermore, the adhesion strength of targeted microbubbles is linearly dependent on the degree of inflammation, suggesting that targeted ultrasound imaging may offer differentiation between various degrees of endothelial dysfunction, and thus detect not only the presence, but also the severity of inflammatory disease processes.     

Internalization of paramagnetic phosphatidylserine-containing liposomes by macrophages

ABSTRACT: BACKGROUND: Inflammation plays an important role in many pathologies, including cardiovascular diseases, neurological conditions and oncology, and is considered an important predictor for disease progression and outcome. In vivo imaging of inflammatory cells will improve diagnosis and provide a read-out for therapy efficacy. Paramagnetic phosphatidylserine (PS)-containing liposomes were developed for magnetic resonance imaging (MRI) and confocal microscopy imaging of macrophages. These nanoparticles also provide a platform to combine imaging with targeted drug delivery. RESULTS: Incorporation of PS into liposomes did not affect liposomal size and morphology up to 12 mol% of PS. Liposomes containing 6 mol% of PS showed the highest uptake by murine macrophages, while only minor uptake was observed in endothelial cells. Uptake of liposomes containing 6 mol% of PS was dependent on the presence of Ca2+ and Mg2+. Furthermore, these 6 mol% PS-containing liposomes were mainly internalized into macrophages, whereas liposomes without PS only bound to the macrophage cell membrane. CONCLUSIONS: Paramagnetic liposomes containing 6 mol% of PS for MR imaging of macrophages have been developed. In vitro these liposomes showed specific internalization by macrophages. Therefore, these liposomes might be suitable for in vivo visualization of macrophage content and for (visualization of) targeted drug delivery to inflammatory cells.     

Polymeric ultrasound contrast agents targeted to integrins: importance of process methods and surface density of ligands

The use of injectable gas-filled microbubbles during ultrasound imaging is accepted as a good method to increase image contrast. Site-targeted microbubbles are expected to provide higher sensitivity and specificity than blood pool contrast agents (CAs). We have shown that covalent attachment of GRGDS peptide fragments to the surface of poly(lactic acid) CAs facilitates attachment to MDA-MB-231 human breast cancer cells in vitro. This paper examines the effect of process conditions during microbubble fabrication and ligand attachment and also changes in ligand surface density and shows that they have important effects on in vitro acoustic response and CA adhesion to breast cancer and cell lines. Use of intermittent sonication in the emulsion step, shortening of reaction times, and increase in freeze-drying times allows for a reduction of 50% in the dose of GRGDS-modified capsules (from 0.16 to 0.012 mg/mL) required to achieve a maximum enhancement of 20 dB; signal loss after 15 min insonation of GRGDS-modified capsules is reduced from a loss of 60% to a loss of 40%, and cell attachment after 10 min contact time is increased from an average of 1.4 +/- 0.86 to 1.8 +/- 0.17 capsules/cell. Optimal attachment is achieved with a molar ratio of total -COOH groups to GRGDS of 1:0.5. The effect of process conditions during microcapsule fabrication, ligand attachment, and ligand surface density on in vitro acoustic response and CA adhesion to breast cancer cell lines in tissue culture are shown to be important parameters that can aid in the future design of an ultrasound CA that allows both cancer detection and treatment, potentially by targeted drug delivery.     

Multifunctional synthetic poly(L-glutamic acid)-based cancer therapeutic and imaging agents

Modern polymer chemistry has led to the generation of a number of biocompatible synthetic polymers that have been increasingly studied as efficient carriers for drugs and imaging agents. Synthetic biocompatible polymers have been used to improve the efficacy of both small-molecular-weight therapeutics and imaging agents. Furthermore, multiple targeted anticancer agents and/or imaging reporters can be attached to a single polymer chain, allowing multifunctional and/or multimodality therapy and molecular imaging. Having both an anticancer drug and an imaging reporter in a single polymer chain allows noninvasive real-time visualization of the pharmacokinetics of polymeric drug delivery systems, which can uncover and explain the complicated mechanisms of in vivo drug delivery and their correlation to pharmacodynamics. This review examines the use of the synthetic biocompatible polymer poly(L-glutamic acid) (PG) as an efficient carrier of cancer therapeutics and imaging agents. This review summarizes and updates our recent research on the use of PG as a platform for drug delivery and molecular imaging, including recent clinical findings with respect to PG-paclitaxel (PG-TXL), the combination of PG-TXL with radiotherapy, mechanisms of action of PG-TXL, and noninvasive visualization of in vivo delivery of polymeric conjugates with contrast-enhanced magnetic resonance imaging, optical imaging, and multimodality imaging.     

Targeted Exosomes for Drug Delivery: Biomanufacturing,Surface Tagging,and Validation

Exosomes hold great potential to deliver therapeutic reagents for cancer treatment due to its inherent low antigenicity. However, several technical barriers, such as low productivity and ineffective cancer targeting, need to be overcome before wide clinical applications. The present study aims at creating a new biomanufacturing platform of cancer‐targeted exosomes for drug delivery. Specifically, a scalable, robust, high‐yield, cell line based exosome production process is created in a stirred‐tank bioreactor, and an efficient surface tagging technique is developed to generate monoclonal antibody (mAb)‐exosomes. The in vitro characterization using transmission electron microscopy, NanoSight, and western blotting confirm the high quality of exosomes. Flow cytometry and confocal laser scanning microscopy demonstrate that mAb‐exosomes have strong surface binding to cancer cells. Furthermore, to validate the targeted drug delivery efficiency, romidepsin, a histone deacetylase inhibitor, is loaded into mAb‐exosomes. The in vitro anti‐cancer toxicity study shows high cytotoxicity of mAb‐exosome‐romidepsin to cancer cells. Finally, the in vivo study using tumor xenograft animal model validates the cancer targeting specificity, anti‐cancer efficacy, and drug delivery capability of the targeted exosomes. In summary, new techniques enabling targeted exosomes for drug delivery are developed to support large‐scale animal studies and to facilitate the translation from research to clinics.     

Cellular imaging of inflammation in atherosclerosis using magnetofluorescent nanomaterials

OBJECTIVE: Magnetofluorescent nanoparticles (MFNPs) offer the ability to image cellular inflammation in vivo. To better understand their cellular targeting and imaging capabilities in atherosclerosis, we investigated prototypical dextran-coated near-infrared fluorescent MFNPs in the apolipoprotein E-deficient (apo E-/-) mouse model. METHODS AND RESULTS: In vitro MFNP uptake was highest in activated murine macrophages (p < .001). Apo E-/- mice (n = 11) were next injected with the MFNP (15 mg/kg iron) or saline. In vivo magnetic resonance imaging (MRI) demonstrated strong plaque enhancement by the MFNPs (p < .001 vs. saline), which was confirmed by multimodality ex vivo MRI and fluorescence reflectance imaging. On fluorescence microscopy, MFNPs were found in cellular-rich areas of atheroma and colocalized with immunofluorescent macrophages over endothelial cells and smooth muscle cells (p < .001). CONCLUSIONS: Here we show that (1) the in vitro and in vivo cellular distribution of atherosclerosis-targeted MFNPs can be quantified by using fluorescence imaging methods; (2) in atherosclerosis, dextranated MFNPs preferentially target macrophages; and (3) MFNP deposition in murine atheroma can be noninvasively detected by in vivo MRI. This study thus provides a foundation for using MFNPs to image genetic and/or pharmacological perturbations of cellular inflammation in experimental atherosclerosis and for the future development of novel targeted nanomaterials for atherosclerosis.     

Long circulating lectin conjugated paclitaxel loaded magnetic nanoparticles: a new theranostic avenue for leukemia therapy

Amongst all leukemias, Bcr-Abl positive chronic myelogenous leukemia (CML) confers resistance to native drug due to multi drug resistance and also resistance to p53 and fas ligand pathways. In the present study, we have investigated the efficacy of microtubule stabilizing paclitaxel loaded magnetic nanoparticles (pac-MNPs) to ascertain its cytotoxic effect on Bcr-Abl positive K562 cells. For active targeted therapy, pac-MNPs were functionalized with lectin glycoprotein which resulted in higher cellular uptake and lower IC(50) value suggesting the efficacy of targeted delivery of paclitaxel. Both pac-MNPs and lectin conjugated pac-MNPs have a prolonged circulation time in serum suggesting increased bioavailability and therapeutics index of paclitaxel in vivo. Further, the molecular mechanism pertaining to pac-induced cytotoxicity was analyzed by studying the involvement of different apoptotic pathway proteins by immunoblotting and quantitative PCR. Our study revealed simultaneous activation of JNK pathway leading to Bcr-Abl instability and the extrinsic apoptotic pathway after pac-MNPs treatment in two Bcr-Abl positive cell lines. In addition, the MRI data suggested the potential application of MNPs as imaging agent. Thus our in vitro and in vivo results strongly suggested the pac-MNPs as a future prospective theranostic tool for leukemia therapy.     

Molecular vehicles for targeted drug delivery

Targeted drug delivery by cell-specific cytokines and antibodies promises greater drug efficacy and reduced side effects. We describe a novel strategy for assembly of drug delivery vehicles that does not require chemical modification of targeting proteins. The strategy relies on a noncovalent binding of standardized "payload" modules to targeting proteins expressed with a "docking" tag. The payload modules are constructed by linking drug carriers to an adapter protein capable of binding to a docking tag. Using fragments of bovine ribonuclease A as an adapter protein and a docking tag, we have constructed vascular endothelial growth factor (VEGF) based vehicles for gene delivery and for liposome delivery. Assembled vehicles displayed remarkable selectivity in drug delivery to cells overexpressing VEGF receptors. We expect that our strategy can be employed for targeted delivery of many therapeutic or imaging agents by different recombinant targeting proteins.     

Local drug and gene delivery through microbubbles and ultrasound

Although gene therapy has great potential as a treatment for diseases, clinical trials are slowed down by the development of a safe and efficient gene delivery system. In this review, we will give an overview of the viral and nonviral vehicles used for drug and gene delivery, and the different nonviral delivery techniques, thereby focusing on delivery through ultrasound contrast agents.The development of ultrasound contrast agents containing encapsulated microbubbles has increased the possibilities not only for diagnostic imaging, but for therapy as well. Microbubbles have been shown to be able to carry drugs and genes, and destruction of the bubbles by ultrasound will result in local release of their contents. Furthermore, ligands can be attached so that they can be targeted to a specific target tissue. The recent advances of microbubbles as vehicles for delivery of drugs and genes will be highlighted.     

Study on the Multidrug Resistance 1 Gene Transfection Efficiency Using Adenovirus Vector Enhanced by Ultrasonic Microbubbles In Vitro

As gene delivery reagents, microbubbles have been successfully used in combination with ultrasound. Shock wave exposure has been shown to transfect cells with naked DNA in vitro, but it has not been tested whether the addition of microbubbles would enhance DNA uptake with adenovirus vector. Therefore, the aim of this study was to study the efficacy and safety of multidrug resistance 1 (MDR1) gene transfer into the bone marrow mononuclear cells of rabbits using adenovirus vector enhanced by ultrasound with microbubbles in vitro. The transfection rate of the MDR1 gene was significantly increased by ultrasound microbubbles with adenovirus. After ultrasonic irradiation, there were transient holes in the cell membrane, which disappeared after irradiation by ultrasound for 24 h. The temporary swelling of the organelles was reversible. Our in vitro findings conclusively demonstrate that the exogenous MDR1 gene transfer into the mononuclear cells of rabbits with adenovirus vector was enhanced by the ultrasonic microbubbles and this transfection technique is safe.     

In vivo detection of phospholipase C by enzyme-activated near-infrared probes

In this article, the characterization of the first near-infrared (NIR) phospholipase-activated molecular beacon is reported, and its utility for in vivo cancer imaging is demonstrated. The probe consists of three elements: a phospholipid (PL) backbone to which the NIR fluorophore, pyropheophorbide a (Pyro), and the NIR Black Hole Quencher 3 (BHQ) were conjugated. Because of the close proximity of BHQ to Pyro, the Pyro-PtdEtn-BHQ probe is self-quenched until enzyme hydrolysis releases the fluorophore. The Pyro-PtdEtn-BHQ probe is highly specific to one isoform of phospholipase C, phosphatidylcholine-specific phospholipase C (PC-PLC), responsible for catabolizing phosphatidylcholine directly to phosphocholine. Incubation of Pyro-PtdEtn-BHQ in vitro with PC-PLC demonstrated a 150-fold increase in fluorescence that could be inhibited by the specific PC-PLC inhibitor tricyclodecan-9-yl xanthogenate (D609) with an IC(50) of 34 ± 8 μM. Since elevations in phosphocholine have been consistently observed by magnetic resonance spectroscopy in a wide array of cancer cells and solid tumors, we assessed the utility of Pyro-PtdEtn-BHQ as a probe for targeted tumor imaging. Injection of Pyro-PtdEtn-BHQ into mice bearing DU145 human prostate tumor xenografts followed by in vivo NIR imaging resulted in a 4-fold increase in tumor radiance over background and a 2 fold increase in the tumor/muscle ratio. Tumor fluorescence enhancement was inhibited with the administration of D609. The ability to image PC-PLC activity in vivo provides a unique and sensitive method of monitoring one of the critical phospholipase signaling pathways activated in cancer, as well as the phospholipase activities that are altered in response to cancer treatment.     

Manufacture of Concentrated,Lipid-based Oxygen Microbubble Emulsions by High Shear Homogenization and Serial Concentration

Gas-filled microbubbles have been developed as ultrasound contrast and drug delivery agents. Microbubbles can be produced by processing surfactants using sonication, mechanical agitation, microfluidic devices, or homogenization. Recently, lipid-based oxygen microbubbles (LOMs) have been designed to deliver oxygen intravenously during medical emergencies, reversing life-threatening hypoxemia, and preventing subsequent organ injury, cardiac arrest, and death. We present methods for scaled-up production of highly oxygenated microbubbles using a closed-loop high-shear homogenizer. The process can produce 2 L of concentrated LOMs (90% by volume) in 90 min. Resulting bubbles have a mean diameter of ~2 μm, and a rheologic profile consistent with that of blood when diluted to 60 volume %. This technique produces LOMs in high capacity and with high oxygen purity, suggesting that this technique may be useful for translational research labs.     

Polymer shelled microparticles for a targeted doxorubicin delivery in cancer therapy

Targeting is a main feature supporting any controlled drug delivery modality. Recently we developed poly(vinyl alcohol), PVA, based microbubbles as a potential new ultrasound contrast agent featuring an efficient ultrasound backscattering and a good shelf stability. The chemical versatility of the polymeric surface of this device offers a vast variety of coupling modalities useful for coating and specific targeting. We have designed a conjugation strategy on PVA shelled microbubbles to enable the localization and the drug delivery on tumor cells by modifying the surface of this polymeric ultrasound contrast agent (UCA) with oxidized hyaluronic acid (HAox). After the conversion of the microbubbles into microcapsules, the kinetics of the release of doxorubicin, a well-known antitumor drug, from uncoated and HAox-coated PVA microbubbles and microcapsules was investigated. Cytocompatibility and bioadhesive properties of the HA-modified microparticles were then tested on the HT-29 tumor cell line. Cytotoxicity to HT-29 tumor cells of microcapsules after loading with doxorubicin was studied, evidencing the efficacy of the HAox coating for the delivery of the drug to cells. These features are a prerequisite for a theranostic, that is, diagnostic and therapeutic, use of polymer-based UCAs.     

Sonoporation: Mechanistic insights and ongoing challenges for gene transfer

Microbubbles first developed as ultrasound contrast agents have been used to assist ultrasound for cellular drug and gene delivery. Their oscillation behavior during ultrasound exposure leads to transient membrane permeability of surrounding cells, facilitating targeted local delivery. The increased cell uptake of extracellular compounds by ultrasound in the presence of microbubbles is attributed to a phenomenon called sonoporation. In this review, we summarize current state of the art concerning microbubble–cell interactions and cellular effects leading to sonoporation and its application for gene delivery. Optimization of sonoporation protocol and composition of microbubbles for gene delivery are discussed.     

Blood oxygenation using microbubble suspensions

Microbubbles have been used in a variety of fields and have unique properties, for example shrinking collapse, long lifetime, efficient gas solubility, a negatively charged surface, and the ability to produce free radicals. In medicine, microbubbles have been used mainly as diagnostic aids to scan various organs of the body, and they have recently been investigated for use in drug and gene delivery. However, there have been no reports of blood oxygenation by use of oxygen microbubble fluids without shell reagents. In this study, we demonstrated that nano or microbubbles can achieve oxygen supersaturation of fluids, and may be sufficiently small and safe for infusion into blood vessels. Although Po(2) increases in fluids resulting from use of microbubbles were inhibited by polar solvents, normal saline solution (NSS) was little affected. Thus, NSS is suitable for production of oxygen-rich fluid. In addition, oxygen microbubble NSS effectively improved hypoxic conditions in blood. Thus, use of oxygen microbubble (nanobubble) fluids is a potentially effective novel method for oxygenation of hypoxic tissues, for infection control, and for anticancer treatment.