Letizia Oddo

Magnetite Nanoparticles Can Be Coupled to Microbubbles to Support Multimodal Imaging

Microbubbles (MBs) are commonly used as injectable ultrasound contrast agent (UCA) in modern ultrasonography. Polymer-shelled UCAs present additional potentialities with respect to marketed lipid-shelled UCAs. They are more robust; that is, they have longer shelf and circulation life, and surface modifications are quite easily accomplished to obtain enhanced targeting and local drug delivery. The next generation of UCAs will be required to support not only ultrasound-based imaging methods but also other complementary diagnostic approaches such as magnetic resonance imaging or computer tomography. This work addresses the features of MBs that could function as contrast agents for both ultrasound and magnetic resonance imaging. The results indicate that the introduction of iron oxide nanoparticles (SPIONs) in the poly(vinyl alcohol) shell or on the external surface of the MBs does not greatly decrease the echogenicity of the host MBs compared with the unmodified one. The presence of SPIONs provides enough magnetic susceptibility to the MBs to accomplish good detectability both in vitro and in vivo. The distribution of SPIONs on the shell and their aggregation state seem to be key factors for the optimization of the transverse relaxation rate.

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.

Targeted doxorubicin delivery by chitosan-galactosylated modified polymer microbubbles to hepatocarcinoma cells

Targeted drug delivery is a main issue in cancer treatment. Taking advantage of recently developed polyvinyl alcohol (PVA)-based microbubbles, which are characterized by chemical versatility of the polymeric surface thereby allowing coating with different ligands, we set up a strategy for the targeted delivery of the anticancer agent doxorubicin to hepatocarcinoma cells. Such microbubbles are exceptionally efficient ultrasound scatterers and thus represent also an option as potential ultrasound contrast agents. Moreover, the oscillation of microbubbles induced by ultrasound could contribute to favor the release of drugs allocated on shell. Specifically, PVA-based microbubbles were reacted with a galactosylated chitosan complex and loaded with doxorubicin to enable the localization and drug delivery to HepG2 hepatocarcinoma cells overexpressing asialoglycoprotein receptors. We demonstrated selectivity and greater bioadhesive properties of the functionalized microbubbles for tumor cells than to normal fibroblasts, which were influenced by the degree of galactosylation. The presence of galactosylated chitosan did not modify the rate of doxorubicin release from microbubbles, whichwas almost complete within 48h. Cellular uptake of doxorubicin loaded on functionalized microbubbles was higher in HepG2 than in normal fibroblasts, which do not over express the asialoglycoprotein receptors. In addition, doxorubicin loaded onto functionalized microbubbles fully retained its cytotoxic activity. Cells were also irradiated with ultrasound, immediately after exposure to microbubbles. An early enhancement of doxorubicin release and cellular drug uptake associated to a concomitant increase in cytotoxicity was observed in HepG2 cells. Overall, results of the study indicate that galactosylated chitosan microbubbles represent promising devices for the targeted delivery of antitumor agents to liver cancer cells.

Multimodality imaging using SPECT/CT and MRI and ligand functionalized 99mTc-labeled magnetic microbubbles

BackgroundIn the present study, we used multimodal imaging to investigate biodistribution in rats after intravenous administration of a new 99mTc-labeled delivery system consisting of polymer-shelled microbubbles (MBs) functionalized with diethylenetriaminepentaacetic acid (DTPA), thiolated poly(methacrylic acid) (PMAA), chitosan, 1,4,7-triacyclononane-1,4,7-triacetic acid (NOTA), NOTA-super paramagnetic iron oxide nanoparticles (SPION), or DTPA-SPION.MethodsExaminations utilizing planar dynamic scintigraphy and hybrid imaging were performed using a commercially available single-photon emission computed tomography (SPECT)/computed tomography (CT) system. For SPION containing MBs, the biodistribution pattern of 99mTc-labeled NOTA-SPION and DTPA-SPION MBs was investigated and co-registered using fusion SPECT/CT and magnetic resonance imaging (MRI). Moreover, to evaluate the biodistribution, organs were removed and radioactivity was measured and calculated as percentage of injected dose.ResultsSPECT/CT and MRI showed that the distribution of 99mTc-labeled ligand-functionalized MBs varied with the type of ligand as well as with the presence of SPION. The highest uptake was observed in the lungs 1 h post injection of 99mTc-labeled DTPA and chitosan MBs, while a similar distribution to the lungs and the liver was seen after the administration of PMAA MBs. The highest counts of 99mTc-labeled NOTA-SPION and DTPA-SPION MBs were observed in the lungs, liver, and kidneys 1 h post injection. The highest counts were observed in the liver, spleen, and kidneys as confirmed by MRI 24 h post injection. Furthermore, the results obtained from organ measurements were in good agreement with those obtained from SPECT/CT.ConclusionsIn conclusion, microbubbles functionalized by different ligands can be labeled with radiotracers and utilized for SPECT/CT imaging, while the incorporation of SPION in MB shells enables imaging using MR. Our investigation revealed that biodistribution may be modified using different ligands. Furthermore, using a single contrast agent with fusion SPECT/CT/MR multimodal imaging enables visualization of functional and anatomical information in one image, thus improving the diagnostic benefit for patients.

Acoustic characterization and contrast imaging of microbubbles encapsulated by polymeric shells coated or filled with magnetic nanoparticles

The combination of superparamagnetic iron oxide nanoparticles with polymeric air-filled microbubbles is used to produce two types of multimodal contrast agents to enhance medical ultrasound and magnetic resonance imaging. The nanoparticles are either covalently linked to the shell or physically entrapped into the shell. In this paper, the characterization of the acoustic properties (backscattered power, fracturing pressure, attenuation and dispersion of the ultrasonic wave) and ultrasound imaging of the two types of magnetic microbubbles are presented. In vitro B-mode images are generated using a medical ultrasound scanner by applying a nonconventional signal processing technique that is suitable to detect polymeric bubbles and based on the combination of multipulse excitation and chirp coding. Even if both types of microbubbles can be considered to be effective ultrasound contrast agents, the different structure of the shell loaded with nanoparticles has a pronounced effect on the echogenicity and the detection sensitivity of the imaging technique. The best results are obtained using microbubbles that are externally coated with nanoparticles. A backscattered power of 20 dB was achieved at lower concentration, and an increment of 8 dB in the contrast-to-tissue ratio was observed with respect to the more rigid microbubbles with particles entrapped into the shell.

Next generation ultrasound platforms for theranostics

Microbubbles are a well-established contrast agent which improves diagnostic ultrasound imaging. During the last decade research has focused on expanding their use to include molecular imaging, targeted therapy and imaging modalities other than ultrasound. However, bioadhesion of targeted microbubbles under physiological flow conditions is still difficult to achieve, the main challenge being connected to the poor stability of lipid microbubbles in the bodys circulation system. In this article, we investigate the use of polymeric microbubbles based on a poly (vinyl alcohol) shell as an alternative to lipid microbubbles. In particular, we report on the development of microbubble shell modification, using mild reaction conditions, with the aim of designing a multifunctional platform to enable diagnosis and therapy. Superparamagnetic iron oxide nanoparticles and a near infrared fluorescent probe, indocyanine green, are coupled to the bubbles surface in order to support magnetic resonance and fluorescence imaging. Furthermore, anchoring cyclic arginyl-glycyl-aspartic acid (RGD) peptide, and cyclodextrin molecules, allows targeting and drug loading, respectively. Last but not least, shell topography is provided by atomic force microscopy. These applications and features, together with the high echogenicity of poly (vinyl alcohol) microbubbles, may offer a more stable alternative to lipid microbubbles for the development of a multimodal theranostic platform.

Visualization of multimodal polymer-shelled contrast agents using ultrasound contrast sequences: an experimental study in a tissue mimicking flow phantom

BackgroundA multimodal polymer-shelled contrast agent (CA) with target specific potential was recently developed and tested for its acoustic properties in a single element transducer setup. Since the developed polymeric CA has different chemical composition than the commercially available CAs, there is an interest to study its acoustic response when using clinical ultrasound systems. The aim of this study was therefore to investigate the acoustic response by studying the visualization capability and shadowing effect of three polymer-shelled CAs when using optimized sequences for contrast imaging.MethodsThe acoustic response of three types of the multimodal CA was evaluated in a tissue mimicking flow phantom setup by measuring contrast to tissue ratio (CTR) and acoustic shadowing using five image sequences optimized for contrast imaging. The measurements were performed over a mechanical index (MI) range of 0.2-1.2 at three CA concentrations (106, 105, 104 microbubbles/ml).ResultsThe CTR-values were found to vary with the applied contrast sequence, MI and CA. The highest CTR-values were obtained when a contrast sequence optimized for higher MI imaging was used. At a CA concentration of 106 microbubbles/ml, acoustic shadowing was observed for all contrast sequences and CAs.ConclusionsThe CAs showed the potential to enhance ultrasound images generated by available contrast sequences. A CA concentration of 106 MBs/ml implies a non-linear relation between MB concentration and image intensity.

Assessment of the viscoelastic and oscillation properties of a nano-engineered multimodality contrast agent.

Combinations of microbubbles (MBs) and superparamagnetic iron oxide nanoparticles (SPIONs) are used to fabricate dual contrast agents for ultrasound and MRI. This study examines the viscoelastic and oscillation characteristics of two MB types that are manufactured with SPIONs and either anchored chemically on the surface (MBs-chem) or physically embedded (MBs-phys) into a polymer shell. A linearized Church model was employed to simultaneously fit attenuation coefficients and phase velocity spectra that were acquired experimentally. The model predicted lower viscoelastic modulus values, undamped resonance frequencies and total damping ratios for MBs-chem. MBs-chem had a resonance frequency of approximately 13 MHz and a damping ratio of approximately 0.9; thus, MBs-chem can potentially be used as a conventional ultrasound contrast agent with the combined functionality of MRI detection. In contrast, MBs-phys had a resonance frequency and damping of 28 MHz and 1.2, respectively, and requires further modification of clinically available contrast pulse sequences to be visualized.

Biological in situ characterization of polymeric microbubble contrast agents

Polymeric microbubbles (MBs) are gas filled particles composed of a thin stabilized polymer shell that have been recently developed as valid contrast agents for the combined use of ultrasonography (US), magnetic resonance imaging (MRI) and single photon emission computer tomography (SPECT) imaging. Due to their buoyancy, the commonly available approaches to study their behaviour in complex media are not easily applicable and their use in modern medicine requires such behaviour to be fully elucidated. Here we have used for the first time flow cytometry as a new high throughput approach that allows characterisation of the MB dispersion, prior to and after exposure in different biological media and we have additionally developed a method that allows characterisation of the strongly bound proteins adsorbed on the MBs, to fully predict their biological behaviour in biological milieu.

Design of Novel Polymer Shelled Ultrasound Contrast Agents: Towards an Ultrasound Triggered Drug Delivery

Ultrasound contrast agents (UCA) have been used for years in the clinical field, for applications such as blood pool enhancement, characterization of liver lesions or perfusion imaging [12, 13, 14, 15, 20]. The contrast agents are generally in the form of spherical voids or cavities filled by a gas, called microbubbles (MB). MBs are stabilized by a coating material such as phospholipids, surfactants, denatured human serum albumin or synthetic polymers. As gas is less dense than liquids or solids, sound travels more slowly in gas than it does in liquid. The difference in the sound speed in the microbubbles creates an acoustic mismatch between tissue and blood surrounding a microbubble, making it an efficient reflector of ultrasound energy. The ability to produce strong signals from microbubbles depends on the stability of the gas particle. Stability has been enhanced by varying the gas composition (air, nitrogen, sulphur hexafluoride, perfluorocarbons) and/or by chemical modification of the microbubble shell [20]. As microbubbles stay in the vascular space and have a behavior similar to red blood cells in the microcirculation, they can be used as intravascular tracers. The possibility to introduce a targeting ligand for a specific interaction with its receptor onto the microbubble shell can be a way to selectively accumulate the contrast agent in the deseased region. Moreover, targeted MBs can be used to carry different drugs to the desired sites and release them after their destruction/cavitation with ultrasound radiation or by chemical/enzymatic cleavage.