Polymer Microbubbles Research Articles

Ultrasound Detection of Myocardial Ischemic Memory Using an E-Selectin Targeting Peptide Amenable to Human Application

Vascular endothelial leukocyte adhesion molecules, such as E-selectin, are acutely upregulated in myocardial ischemia/reperfusion and are thus "ischemic memory" biomarkers for recent cardiac ischemia. We sought to develop an ultrasound molecular imaging agent composed of microbubbles (MBs) targeted to E-selectin to enable the differential diagnosis of myocardial ischemia in patients presenting with chest pain of unclear etiology. Biodegradable polymer MBs were prepared bearing a peptide with specific human E-selectin affinity (MBESEL). Control MBs had scrambled peptide (MBCTL) or nonspecific IgG (MBIgG). MBESEL adhesion to activated rat endothelial cells (ECs) was confirmed in vitro in a flow system and in vivo with intravital microscopy of rat cremaster microcirculation. Ultrasound molecular imaging of recent myocardial ischemia was performed in rats 4 hours after transient (15 minutes) coronary occlusion. MBESEL adhesion was higher to inflamed versus normal ECs in vitro; there was no difference in MBCTL or MBIgG adhesion to inflamed versus normal ECs. There was greater adhesion of MBESEL to inflamed versus noninflamed microcirculation and minimal adhesion of MBCTL or MBIgG under any condition. Ultrasound imaging after injection of MBSEL demonstrated persistent contrast enhancement of the previously ischemic region. Videointensity in postischemic myocardium after MBESEL was higher than that in the nonischemic bed (11.6 ± 2.7 dB vs 3.6 ± 0.8 dB, p < .02) and higher than that after MBCTL (4.0 ± 1.0 dB, p < .03) or MBIgG (1.7 ± 0.1 dB, p < .03). MBs targeted to E-selectin via a short synthetic peptide with human E-selectin binding affinity enables echocardiographic detection of recent ischemia, setting the stage for clinical myocardial ischemic memory imaging to identify acute coronary syndromes.

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.

In Vitro Gene Delivery with Ultrasound-Triggered Polymer Microbubbles

In the work described here, gene delivery using polymer microbubbles triggered by ultrasound in vitro was investigated. The effects of pressure amplitude (0–2 MPa), center frequency (1–5 MHz), pulse length (3–12,000 μs), pulse repetition frequency (5–20,000 Hz) and exposure time (0–30 s) on transfection efficiency and cell viability were examined. The effects of radiation force, calcium ion concentration and timing of treatments were also examined. Cells were successfully transfected with pressure amplitudes as low as 250 kPa. Transfection was most efficient at lower frequencies and longer pulse lengths, with a transfection efficiency of 24.2 ± 2.0% achieved using a center frequency of 1 MHz, pressure amplitude of 1 MPa, pulse length of 12,000 μs and pulse repetition frequency of 5 Hz. Gene delivery was also affected by the extracellular calcium ion concentration and the timing of treatments.

Preparation and characterization of QDs-loaded PLGA microbubbles as fluorescent-ultrasonic dual-modality imaging agent

Objective To prepare the quantum dots(QDs) (CdTe-MPA)-loaded polymer(lactic-coglycolic acid,PLGA) microbubbles(MBQDs@PLGA) as dual-modality imaging agent for both fluorescent and ultrasonic imaging ability.Methods The MBQDs@PLGA were generated by the double emulsion technique,then filling in C3F8 after freeze-drying.Confocal laser scanning microscope(CLSM) and transmission electron microscope(TEM) were used to confirm the load of quantum dots in the MBs.Fluorospectro photometer spectra of the MBQDs@PLGA were analyzed to demonstrate the fluorescent imaging ability and determine the encapsulation efficiency by using the regression equation.Imaging experiments was applied to validate the fluorescent and ultrasonic imaging ability of the MBQDs@PLGA both by imaging of the model in vitro and by imaging of ovarian tumor blood vessels of tumor-bearing nude mouse in vivo.Results At excitation 272 nm the MBQDs@PLGA peak of the emission spectrum was 549 nm,and the encapsulation efficiency was 54％.The average diameter of MBQDs@PLGA was (1.7 ±0.2)μm,CLSM and TEM results confirmed the QDs-loaded in MBQDs-PLGA.The imaging results of MBQDs@PLGA showed a dual-modality imaging ability both fluorescent and ultrasonic imaging.Conclusions MBQDs@PLGA present fluorescence-ultrasound dual mode imaging performance by the QDs embedding in polymer microbubbles,and explore a new development train of thought of multi-mode imaging agent. Key words: Ultrasonography; Microbubbles; Lactic-co-glycolic acid; Quantum dots; Dual modality imaging

Doxorubicin loaded superparamagnetic PLGA-iron oxide multifunctional microbubbles for dual-mode US/MR imaging and therapy of metastasis in lymph nodes

Current strategies for tumor-induced sentinel lymph node detection and metastasis therapy have limitations. In this work, we co-encapsulated iron oxide nanoparticles and chemotherapeutic drug into poly(lactic-co-glycolic acid) (PLGA) microbubbles to form multifunctional polymer microbubbles (MPMBs) for both tumor lymph node imaging and therapy. Fe3O4 nanoparticles and doxorubicin (DOX) co-encapsulated PLGA microbubbles were prepared and filled with perfluorocarbon gas. Enhancement of ultrasound (US)/magnetic resonance (MR) imaging and US triggered drug delivery were evaluated both in vitro and in vivo. The MPMBs exhibited characters like narrow size distribution and smooth surface with a mean diameter of 868.0 ± 68.73 nm. In addition, varying the concentration of Fe3O4 nanoparticles in the bubbles did not significantly influence the DOX encapsulation efficiency or drug loading efficiency. Our in vitro results demonstrated that these MPMBs could enhance both US and MR imaging which was further validated in vivo showing that these MPMBs enhanced tumor lymph nodes signals. The anti-tumor effect of MPMBs mediated chemotherapy was assessed in vivo using end markers like tumor proliferation index, micro blood vessel density and micro lymphatic vessel density, which were shown consistently the lowest after the MPMBs plus sonication treatment compared to controls. In line with these findings, the tumor cell apoptotic index was found the largest after the MPMBs plus sonication treatment. In conclusion, we have successfully developed a doxorubicin loaded superparamagnetic PLGA-Iron Oxide multifunctional theranostic agent for dual-mode US/MR Imaging of lymph node, and for low frequency US triggered therapy of metastasis in lymph nodes, which might provide a strategy for the imaging and chemotherapy of primary tumor and their metastases.

Preparation of polymer microbubbles encapsulated iron oxide nanopraticles and doxorubicin and characterization in vitro

Objective To prepare a kind of superparamagnetic iron oxide (SPIO) and doxorubicin loaded multifunctional polymer microbubbles (MPMBs),and to explore its potential application as an ultrasound(US)/magnetic resonance (MR) imaging contrast agent in vitro.Methods The MPMBs and normal polymer microbubbles (PMBs) were made by double emulsion and freeze-drying methods.The physical property,drug encapsulation efficiency and the drug-loading efficiency of MPMBs were determined,and the release property and US/MR imaging enhancement of MPMBs were observed.Results The MPMBs had a regular shape and narrow size distribution.The drug encapsulation efficiency was (60.20±2.69) ％,and the drug-loading efficiency was (6.02 ± 0.27) ％.The in vitro release experiment showed that ultrasound can promote the release of doxorubicin in MPMBs.US imaging in vitro showed that the enhancement of MPMBs was better than PMBs,and MR imaging in vitro conformed that MPMBs could well enhance MR imaging.Conclusions The MPMBs is a multifunctional contrast agent with the treatment function as well as US/MR dual-mode imaging enhancement effect. Key words: Microbubbles ; Iron oxide ; Doxorubicin ; Polymer

Abstract 19653: Therapeutic Potential of Long Ultrasound Tone Bursts in Microbubble-Ultrasound Mediated Therapeutics: Mechanistic Insights Using High Speed Imaging

Background: Microbubble (MB)-Ultrasound (US) assisted therapy has been shown to restore perfusion in myocardial infarction (sonothrombolysis). Due to a presumption that MBs under high acoustic pressure do not persist after long acoustic cycles, most schemes of US-assisted therapy utilize a very short US pulse (a few acoustic cycles) when a high US pressure is used. However, we have recently observed enhanced in vitro sonothrombolytic effect using MBs and very long US pulses, at high acoustic pressures. We therefore sought to explore the actual fate of MBs during a long acoustic cycle exposure by visual observation of MB acoustic behaviors at varying pulse lengths. Methods: MB behaviors were optically observed during long US tone burst excitation using an ultra-high speed imaging system that allows microscopic visualization of MB cavitation. The system is capable of capturing 128 frames at up to 25 million frames per second (Mfps). Long US tone bursts (up to 5,000 acoustic cycles) at 1 MHz and various pressures (0.25, 0.5, 1.0, and 1.5 MPa) were used to study the dynamic behavior of both lipid and polymer MBs in a 200-µm diameter cellulose tubing. Results: High speed movies of the MBs during long tone burst excitation showed that MBs first underwent inertial cavitation then formed gas-filled aggregates that continued to oscillate. The locations of the aggregates were random due to the dynamic nature of MB destruction. Figure 1 shows still frames of a high speed movie (5 Mpfs) of polymer MBs 1,000 acoustic cycles into a long tone burst at 1.5 MPa: MB aggregates continued to oscillate, break up, and new aggregates were formed. Conclusion: These data show for the first time that MBs survive as aggregates that continue to oscillate with large amplitude during a long tone burst, demonstrating the therapeutic potential for US-assisted therapy. This discovery suggests that long acoustic cycles have additional therapeutic effect for MB-US assisted therapies such as sonothrombolysis.

Focused ultrasound and microbubbles for enhanced extravasation

The permeability of blood vessels for albumin can be altered by using ultrasound and polymer or lipid-shelled microbubbles. The region in which the microbubbles were destroyed with focused ultrasound was quantified in gel phantoms as a function of pressure, number of cycles and type of microbubble. At 2MPa the destruction took place in a fairly wide area for a lipid-shelled agent, while for polymer-shelled agents at this setting, distinct destruction spots with a radius of only 1mm were obtained. When microbubbles with a thicker shell were used, the pressure above which the bubbles were destroyed shifts to higher values. In vivo both lipid and polymer microbubbles increased the extravasation of the albumin binding dye Evans Blue, especially in muscle leading to about 6–8% of the injected dose to extravasate per gram muscle tissue 30min after start of the treatment, while no Evans Blue could be detected in muscle in the absence of microbubbles. Variation in the time between ultrasound treatment and Evans Blue injection, demonstrated that the time window for promoting extravasation is at least an hour at the settings used. In MC38 tumors, extravasation already occurred without ultrasound and only a trend towards enhancement with about a factor of 2 could be established with a maximum percentage injected dose per gram of 3%. Ultrasound mediated microbubble destruction especially enhances the extravasation in the highly vascularized outer part of the MC38 tumor and adjacent muscle and would, therefore, be most useful for release of, for instance, anti-angiogenic drugs.

Abstract 1065: A New Method for Stem Cell Imaging Using Contrast Ultrasound

Background : Mesenchymal stem cells (MSCs) are a promising new cardiac reparative therapy. The distribution of delivered MSCs is poorly understood due to limited methods to track them in vivo . To test the hypothesis that ultrasound can detect MSCs, gas-filled microbubbles (MBs) were developed for MSC uptake. Methods : Perfluorocarbon gas-filled MBs were synthesized from a biodegradable polymer. Cultured rat and human bone-marrow derived MSCs were dwelled with MBs (12 hrs), washed, and suspended in medium. MSC uptake of MBs and MB retention of gas were confirmed with transmission electron microscopy (TEM) and density centrifugation. Spectral analysis of free MB response to up to 10MHz ultrasound was performed. 2nd harmonic and contrast pulse sequence (CPS) 2D echo imaging (7MHz) of MB-labeled and unlabeled MSCs was performed at a mechanical index (MI) 0.3 and 1.9. Videointensity was measured in 200 frames/capture. Results: Frequency spectra of free MBs contained 2nd and 3rd harmonics, indicating MB non-linearity. TEM showed MBs within MSCs (Figure ). By density centrifugation, MSCs with MBs were less dense than MSCs alone, confirming intracellular MB gas retention. Videointensity (gray scale units) of MSCs containing MBs vs MSCs alone was higher at both low MI (35±2 vs 0±0 harmonic, p&lt;0.005; 21±2 vs 15±0 CPS, p&lt;0.005), and high MI (14±2 vs 6±1 harmonic, p&lt;0.005; 6±2 vs 0±0 CPS, p&lt;0.005). Conclusions : Our acoustically active polymer MBs are internalized by MSCs and render them detectable with ultrasound. Because the polymer degrades slowly, this approach may permit serial non-invasive in vivo ultrasound imaging of MSC fate, thus facilitating optimization of cell therapy strategies.

Kinematically Stabilized Microbubble Actuator Arrays

In this paper, the concept, fabrication, and characterization of a kinematically stabilized polymeric microbubble actuator (endoskeletal microbubble actuator) for a pneumatic tactile display application are presented. The kinematic stabilization is achieved by the combination of two polymeric layers with complementary functions: a microcorrugated parylene diaphragm layer as a ldquoskeletonrdquo to provide a directional deflection in a desired axial direction while suppressing undesired lateral deflections and an overcoated elastomer diaphragm layer as a ldquoskinrdquo to help the extended membrane recoil to its original shape, ensuring diaphragm stability. Arrays of microcorrugated diaphragms are implemented in a mass producible fashion using inclined rotational UV lithography, micromolding, and pattern transfer techniques. Both the number of corrugations and the corrugation profile of the endoskeletal actuator are determined through numerical analysis, taking into account the constraints of the microfabrication processes utilized. A prototype of a single endoskeletal bubble actuator with a diameter of 2.6 mm has been fabricated and characterized. For comparison purposes, elastomer microcorrugated diaphragm (skin only) actuators and parylene microcorrugated diaphragm (skeleton only) actuators of the same materials and dimensions have also been fabricated and tested. While the skin-only diaphragm actuators demonstrated undesired omnidirectional inflation and the skeleton-only diaphragm actuators have shown unstable and irreversible deformation during extension, the proposed endoskeletal microbubble actuators have shown stable reversible axial extensions with a deflection of approximately 0.9 mm. A 6 6 array of endoskeletal polymer microbubble actuators integrated with a microfluidic manifold has been successfully fabricated, demonstrating its mass manufacturability.

Tethering Functional Ligands onto Shell of Ultrasound Active Polymeric Microbubbles

Hollow (air-filled) microparticles, i.e., microbubbles, provide a promising novel vehicle for both local delivery of therapeutic agents and simultaneous diagnostic ultrasound echo investigations. In this paper, we describe the synthetic routes for decorating the polymeric shell of a poly(vinyl alcohol)-based microbubble with low and high molecular weight ligands with pharmacological relevance. Investigations on physical properties of microbubbles and surface chemical coupling with different cargo molecules such as L-cysteine, L-lysine, poly(L-lysine), chitosan, and beta-cyclodextrin were carried out by CD and NMR spectroscopies, confocal laser scanning microscopy, and microcalorimetry. The in vitro cytotoxicity and biocompatibility of the polymer microbubbles have been also determined toward different cell lines. The results are discussed in terms of the features shown by this device, i.e., injectability, long shelf life, ease of preparation, biocompatibility, loading and cargo capacities, and functional properties.

Reelin MRNA is decreased in neurons of the hippocampus and temporal cortex in schizophrenia: A cellular in situ hybridisation study

Current strategies for tumor-induced sentinel lymph node detection and metastasis therapy have limitations. In this work, we co-encapsulated iron oxide nanoparticles and chemotherapeutic drug into poly(lactic-co-glycolic acid) (PLGA) microbubbles to form multifunctional polymer microbubbles (MPMBs) for both tumor lymph node imaging and therapy. Fe3O4 nanoparticles and doxorubicin (DOX) co-encapsulated PLGA microbubbles were prepared and filled with perfluorocarbon gas. Enhancement of ultrasound (US)/magnetic resonance (MR) imaging and US triggered drug delivery were evaluated both in vitro and in vivo. The MPMBs exhibited characters like narrow size distribution and smooth surface with a mean diameter of 868.0 ± 68.73 nm. In addition, varying the concentration of Fe3O4 nanoparticles in the bubbles did not significantly influence the DOX encapsulation efficiency or drug loading efficiency. Our in vitro results demonstrated that these MPMBs could enhance both US and MR imaging which was further validated in vivo showing that these MPMBs enhanced tumor lymph nodes signals. The anti-tumor effect of MPMBs mediated chemotherapy was assessed in vivo using end markers like tumor proliferation index, micro blood vessel density and micro lymphatic vessel density, which were shown consistently the lowest after the MPMBs plus sonication treatment compared to controls. In line with these findings, the tumor cell apoptotic index was found the largest after the MPMBs plus sonication treatment. In conclusion, we have successfully developed a doxorubicin loaded superparamagnetic PLGA-Iron Oxide multifunctional theranostic agent for dual-mode US/MR Imaging of lymph node, and for low frequency US triggered therapy of metastasis in lymph nodes, which might provide a strategy for the imaging and chemotherapy of primary tumor and their metastases.