Ultrasound enhanced siRNA delivery using cationic liposome-microbubble complexes for the treatment of squamous cell carcinoma.

Rationale: Microbubble (MB) contrast agents combined with ultrasound targeted microbubble cavitation (UTMC) are a promising platform for site-specific therapeutic oligonucleotide delivery. We investigated UTMC-mediated delivery of siRNA directed against epidermal growth factor receptor (EGFR), to squamous cell carcinoma (SCC) via a novel MB-liposome complex (LPX). Methods: LPXs were constructed by conjugation of cationic liposomes to the surface of C4F10 gas-filled lipid MBs using biotin/avidin chemistry, then loaded with siRNA via electrostatic interaction. Luciferase-expressing SCC-VII cells (SCC-VII-Luc) were cultured in Petri dishes. The Petri dishes were filled with media in which LPXs loaded with siRNA against firefly luciferase (Luc siRNA) were suspended. Ultrasound (US) (1 MHz, 100-µs pulse, 10% duty cycle) was delivered to the dishes for 10 sec at varying acoustic pressures and luciferase assay was performed 24 hr later. In vivo siRNA delivery was studied in SCC-VII tumor-bearing mice intravenously infused with a 0.5 mL saline suspension of EGFR siRNA LPX (7×108 LPX, ~30 µg siRNA) for 20 min during concurrent US (1 MHz, 0.5 MPa spatial peak temporal peak negative pressure, five 100-µs pulses every 1 ms; each pulse train repeated every 2 sec to allow reperfusion of LPX into the tumor). Mice were sacrificed 2 days post treatment and tumor EGFR expression was measured (Western blot). Other mice (n=23) received either EGFR siRNA-loaded LPX + UTMC or negative control (NC) siRNA-loaded LPX + UTMC on days 0 and 3, or no treatment ("sham"). Tumor volume was serially measured by high-resolution 3D US imaging. Results: Luc siRNA LPX + UTMC caused significant luciferase knockdown vs. no treatment control, p<0.05) in SCC-VII-Luc cells at acoustic pressures 0.25 MPa to 0.9 MPa, while no significant silencing effect was seen at lower pressure (0.125 MPa). In vivo, EGFR siRNA LPX + UTMC reduced tumor EGFR expression by ~30% and significantly inhibited tumor growth by day 9 (~40% decrease in tumor volume vs. NC siRNA LPX + UTMC, p<0.05). Conclusions: Luc siRNA LPXs + UTMC achieved functional delivery of Luc siRNA to SCC-VII-Luc cells in vitro. EGFR siRNA LPX + UTMC inhibited tumor growth and suppressed EGFR expression in vivo, suggesting that this platform holds promise for non-invasive, image-guided targeted delivery of therapeutic siRNA for cancer treatment.

A multicellular brain spheroid model for studying the mechanisms and bioeffects of ultrasound-enhanced drug penetration beyond the blood‒brain barrier.

The blood‒brain barrier (BBB) acts as a hindrance to drug therapy reaching the brain. With an increasing incidence of neurovascular diseases and brain cancer metastases, there is a need for an ideal in vitro model to develop novel methodologies for enhancing drug delivery to the brain. Here, we established a multicellular human brain spheroid model that mimics the BBB both architecturally and functionally. Within the spheroids, endothelial cells and pericytes localized to the periphery, while neurons, astrocytes, and microglia were distributed throughout. Ultrasound-targeted microbubble cavitation (UTMC) is a novel noninvasive technology for enhancing endothelial drug permeability. We utilized our three-dimensional (3D) model to study the feasibility and mechanisms regulating UTMC-induced hyperpermeability. UTMC caused a significant increase in the penetration of 10 kDa Texas red dextran (TRD) into the spheroids, 100 µm beyond the BBB, without compromising cell viability. This hyperpermeability was dependent on UTMC-induced calcium (Ca2+) influx and endothelial nitric oxide synthase (eNOS) activation. Our 3D brain spheroid model, with its intact and functional BBB, offers a valuable platform for studying the bioeffects of UTMC, including effects occurring spatially distant from the endothelial barrier.

STAT3 decoy oligonucleotide-carrying microbubbles with pulsed ultrasound for enhanced therapeutic effect in head and neck tumors.

Signal transducer and activator of transcription-3 (STAT3) is an oncogenic transcription factor implicated in carcinogenesis, tumor progression, and drug resistance in head and neck squamous cell carcinoma (HNSCC). A decoy oligonucleotide targeting STAT3 offers a promising anti-tumor strategy, but achieving targeted tumor delivery of the decoy with systemic administration poses a significant challenge. We previously showed the potential for STAT3 decoy-loaded microbubbles, in conjunction with ultrasound targeted microbubble cavitation (UTMC), to decrease tumor growth in murine squamous cell carcinoma. As a next step towards clinical translation, we sought to determine the anti-tumor efficacy of our STAT3 decoy delivery platform against human HNSCC and the effect of higher STAT3 decoy microbubble loading on tumor cell inhibition. STAT3 decoy was loaded on cationic lipid microbubbles (STAT3-MB) or loaded on liposome-conjugated lipid microbubbles to form STAT3-loaded liposome-microbubble complexes (STAT3-LPX). UTMC treatment efficacy with these two formulations was evaluated in vitro using viability and apoptosis assays in CAL33 (human HNSCC) cells. Anti-cancer efficacy in vivo was performed in a CAL33 tumor murine xenograft model. UTMC with STAT3-MB caused significantly lower CAL33 cell viability compared to UTMC with STAT3-LPX (56.8±8.4% vs 84.5±8.8%, respectively, p<0.05). In vivo, UTMC with STAT3-MB had strong anti-tumor effects, with significantly less tumor burden and greater survival compared to that of UTMC with microbubbles loaded with a mutant control decoy and untreated control groups (p<0.05). UTMC with STAT3 decoy-loaded microbubbles significantly decreases human HNSSC tumor progression. These data set the stage for clinical translation of our microbubble platform as an imaged-guided, targeted delivery strategy for STAT3 decoy, or other nucleotide-based therapeutics, in human cancer treatment.

Heme Induces IL-6 and Cardiac Hypertrophy Genes Transcripts in Sickle Cell Mice.

Emerging data indicate that free heme promotes inflammation in many different disease settings, including in sickle cell disease (SCD). Although free heme, proinflammatory cytokines, and cardiac hypertrophy are co-existing features of SCD, no mechanistic links between these features have been demonstrated. We now report significantly higher levels of IL-6 mRNA and protein in hearts of the Townes sickle cell disease (SS) mice (2.9-fold, p ≤ 0.05) than control mice expressing normal human hemoglobin (AA). We find that experimental administration of heme 50 µmoles/kg body weight induces IL-6 expression directly in vivo and induces gene expression markers of cardiac hypertrophy in SS mice. We administered heme intravenously and found that within three hours plasma IL-6 protein significantly increased in SS mice compared to AA mice (3248 ± 275 vs. 2384 ± 255 pg/ml, p ≤ 0.05). In the heart, heme induced a 15-fold increase in IL-6 transcript in SS mice heart compared to controls. Heme simultaneously induced other markers of cardiac stress and hypertrophy, including atrial natriuretic factor (Nppa; 14-fold, p ≤ 0.05) and beta myosin heavy chain (Myh7; 8-fold, p ≤ 0.05) in SS mice. Our experiments in Nrf2-deficient mice indicate that the cardiac IL-6 response to heme does not require Nrf2, the usual mediator of transcriptional response to heme for heme detoxification by heme oxygenase-1. These data are the first to show heme-induced IL-6 expression in vivo, suggesting that hemolysis may play a role in the elevated IL-6 and cardiac hypertrophy seen in patients and mice with SCD. Our results align with published evidence from rodents and humans without SCD that suggest a causal relationship between IL-6 and cardiac hypertrophy.

Mechanistic Insight into Sonoporation with Ultrasound-Stimulated Polymer Microbubbles.

Sonoporation is emerging as a feasible, non-viral gene delivery platform for the treatment of cardiovascular disease and cancer. Despite promising results, this approach remains less efficient than viral methods. The objective of this work is to help substantiate the merit of polymeric microbubble sonoporation as a non-viral, localized cell permeation and payload delivery strategy by taking a ground-up approach to elucidating the fundamental mechanisms at play. In this study, we apply simultaneous microscopy of polymeric microbubble sonoporation over its intrinsic biophysical timescales-with sub-microsecond resolution to examine microbubble cavitation and millisecond resolution over several minutes to examine local macromolecule uptake through enhanced endothelial cell membrane permeability-bridging over six orders of magnitude in time. We quantified microbubble behavior and resulting sonoporation thresholds at transmit frequencies of 0.5, 1 and 2 MHz, and determined that sonic cracking is a necessary but insufficient condition to induce sonoporation. Further, sonoporation propensity increases with the extent of sonic cracking, namely, from partial to complete gas escape from the polymeric encapsulation. For the subset that exhibited complete gas escape from sonic cracking, a proportional relationship between the maximum projected gas area and resulting macromolecule uptake was observed. These results have revealed one aspect of polymeric bubble activity on the microsecond time scale that is associated with eliciting sonoporation in adjacent endothelial cells, and contributes toward an understanding of the physical rationale for sonoporation with polymer-encapsulated microbubble contrast agents.

Ultrasound Molecular Imaging of Angiogenesis Using Vascular Endothelial Growth Factor-Conjugated Microbubbles.

Imaging of angiogenesis receptors could provide a sensitive and clinically useful method for detecting neovascularization such as occurs in malignant tumors, and responses to antiangiogenic therapies for such tumors. We tested the hypothesis that microbubbles (MB) tagged with human VEGF121 (MBVEGF) bind to the kinase insert domain receptor (KDR) in vitro and angiogenic endothelium in vivo, and that this specific binding can be imaged on a clinical ultrasound system. In this work, targeted adhesion of MBVEGF was evaluated in vitro using a parallel plate flow system containing adsorbed recombinant human KDR. There was more adhesion of MBVEGF to KDR-coated plates when the amount of VEGF121 on each MB or KDR density on the plate was increased. MBVEGF adhesion to KDR-coated plates decreased with increasing wall shear rate. On intravital microscopic imaging of bFGF-stimulated rat cremaster muscle, there was greater microvascular adhesion of MBVEGF compared to that of isotype IgG-conjugated control MB (MBCTL). To determine if MBVEGF could be used to ultrasonically image angiogenesis, ultrasound imaging was performed in mice bearing squamous cell carcinoma after intravenous injection of MBVEGF. Ultrasound videointensity enhancement in tumor was significantly higher for MBVEGF (17.3 ± 9.7 dB) compared to MBCTL (3.8 ± 4.4 dB, n = 6, p < 0.05). This work demonstrates the feasibility of targeted ultrasound imaging of an angiogenic marker using MBVEGF. This approach offers a noninvasive bedside method for detecting tumor angiogenesis and could be extended to other applications such as molecular monitoring of therapeutic angiogenesis or antiangiogenic therapies in cardiovascular disease or cancer.

Biophysical insight into mechanisms of sonoporation.

This study presents a unique approach to understanding the biophysical mechanisms of ultrasound-triggered cell membrane disruption (i.e., sonoporation). We report direct correlations between ultrasound-stimulated encapsulated microbubble oscillation physics and the resulting cellular membrane permeability by simultaneous microscopy of these two processes over their intrinsic physical timescales (microseconds for microbubble dynamics and seconds to minutes for local macromolecule uptake and cell membrane reorganization). We show that there exists a microbubble oscillation-induced shear-stress threshold, on the order of kilopascals, beyond which endothelial cellular membrane permeability increases. The shear-stress threshold exhibits an inverse square-root relation to the number of oscillation cycles and an approximately linear dependence on ultrasound frequency from 0.5 to 2 MHz. Further, via real-time 3D confocal microscopy measurements, our data provide evidence that a sonoporation event directly results in the immediate generation of membrane pores through both apical and basal cell membrane layers that reseal along their lateral area (resealing time of ∼<2 min). Finally, we demonstrate the potential for sonoporation to indirectly initiate prolonged, intercellular gaps between adjacent, confluent cells (∼>30-60 min). This real-time microscopic approach has provided insight into both the physical, cavitation-based mechanisms of sonoporation and the biophysical, cell-membrane-based mechanisms by which microbubble acoustic behaviors cause acute and sustained enhancement of cellular and vascular permeability.

Dynamic Behavior of Microbubbles during Long Ultrasound Tone-Burst Excitation: Mechanistic Insights into Ultrasound-Microbubble Mediated Therapeutics Using High-Speed Imaging and Cavitation Detection.

Ultrasound (US)-microbubble (MB)-mediated therapies have been found to restore perfusion and enhance drug/gene delivery. On the presumption that MBs do not persist during long US exposure under high acoustic pressures, most schemes use short US pulses when a high US pressure is employed. However, we recently observed an enhanced thrombolytic effect using long US pulses at high acoustic pressures. Therefore, we explored the fate of MBs during long tone-burst exposures (5 ms) at various acoustic pressures and MB concentrations via direct high-speed optical observation and passive cavitation detection. MBs first underwent stable or inertial cavitation depending on the acoustic pressure and then formed gas-filled clusters that continued to oscillate, break up and form new clusters. Cavitation detection confirmed continued, albeit diminishing, acoustic activity throughout the 5-ms US excitation. These data suggest that persisting cavitation activity during long tone bursts may confer additional therapeutic effects.

Low Intensity Ultrasound Mediated Liposomal Doxorubicin Delivery Using Polymer Microbubbles.

Cardiotoxicity is the major dose-limiting factor in the chemotherapeutic use of doxorubicin (Dox). A delivery vehicle that can be triggered to release its payload in the tumoral microvasculature but not in healthy tissue would help improve the therapeutic window of the drug. Delivery strategies combining liposomal encapsulated Dox (LDox), microbubbles (MBs), and ultrasound (US) have been shown to improve therapeutic efficacy of LDox, but much remains to be known about the mechanisms and the US conditions that maximize cytotoxicity using this approach. In this study, we compared different US pulses in terms of drug release and acute toxicity. Drug uptake and proliferation rates using low-intensity US were measured in squamous cell carcinoma cells exposed to LDox conjugated to or coinjected with polymer MBs. The aims of this study were: (1) to compare the effects of low- and high-pressure US on Dox release kinetics; (2) to evaluate whether conjugating the liposome to the MB surface (DoxLPX) is an important factor for drug release and cytotoxicity; and (3) to determine which US parameters most inhibit cell proliferation and whether this inhibition is mediated by drug release or the MB/US interaction with cells. Low-pressure US (170 kPa) at high duty cycle (stable cavitation) released up to ∼ 70% of the encapsulated Dox from the DoxLPX, thus improving Dox bioavailability and cellular uptake and leading to a significant reduction in cell proliferation at 48 h. Flow cytometry showed that US generating stable oscillations of DoxLPX significantly increased cellular Dox uptake at 4 h after US exposure compared to LDox. Drug uptake was correlated with cytotoxicity at 48 h. Our results demonstrate that Dox-containing liposomes conjugated to polymer MBs can be triggered to release ∼ 70% of their payload using noninertial US. Following release, Dox became bioavailable to the cells and induced significantly higher cytotoxicity compared to nonreleased encapsulated drug. Our findings show promise for targeted drug delivery using this theranostic delivery platform at low US intensities.

Ultrasound Targeted Microbubble Destruction-Mediated Delivery of a Transcription Factor Decoy Inhibits STAT3 Signaling and Tumor Growth.

Signal transducer and activator of transcription 3 (STAT3) is constitutively activated in many cancers where it acts to promote tumor progression. A STAT3-specific transcription factor decoy has been developed to suppress STAT3 downstream signaling, but a delivery strategy is needed to improve clinical translation. Ultrasound-targeted microbubble destruction (UTMD) has been shown to enhance image-guided local delivery of molecular therapeutics to a target site. The objective of this study was to deliver STAT3 decoy to squamous cell carcinoma (SCC) tumors using UTMD to disrupt STAT3 signaling and inhibit tumor growth. Studies performed demonstrated that UTMD treatment with STAT3 decoy-loaded microbubbles inhibited STAT3 signaling in SCC cells in vitro. Studies performed in vivo demonstrated that UTMD treatment with STAT3 decoy-loaded microbubbles induced significant tumor growth inhibition (31-51% reduced tumor volume vs. controls, p < 0.05) in mice bearing SCC tumors. Furthermore, expression of STAT3 downstream target genes (Bcl-xL and cyclin D1) was significantly reduced (34-39%, p < 0.05) in tumors receiving UTMD treatment with STAT3 decoy-loaded microbubbles compared to controls. In addition, the quantity of radiolabeled STAT3 decoy detected in tumors eight hours after treatment was significantly higher with UTMD treatment compared to controls (70-150%, p < 0.05). This study demonstrates that UTMD can increase delivery of a transcription factor decoy to tumors in vivo and that the decoy can inhibit STAT3 signaling and tumor growth. These results suggest that UTMD treatment holds potential for clinical use to increase the concentration of a transcription factor signaling inhibitor in the tumor.

Suspension-Expansion of Bone Marrow Results in Small Mesenchymal Stem Cells Exhibiting Increased Transpulmonary Passage Following Intravenous Administration.

Mesenchymal stem cells (MSCs) have been extensively explored in a variety of regenerative medicine applications. The relatively large size of MSCs expanded in tissue culture flasks leads to retention in the microcirculation of the lungs following intravenous delivery, reducing their capacity to reach target sites. We explored whether the expansion of whole marrow in suspension cultures would yield smaller MSCs with increased capacity to traverse the pulmonary microcirculation compared with traditional monolayer cultures. We tested this hypothesis using rat marrow in a suspension bioreactor culture with fibronectin-coated microcarriers, leading to sustained expansion of both the microbead-adherent cells, as well as of a nonadherent cell fraction. Magnetic depletion of CD45(+) cells from the bioreactor cultures after 5 weeks led to a highly enriched CD73(+)/CD90(+)/CD105(+) MSC population. The bioreactor-grown MSCs were significantly smaller than parallel monolayer MSCs (15.1 ± 0.9 µm vs. 18.5 ± 2.3 µm diameter, p<0.05). When fluorescently labeled bioreactor-grown MSCs were intravenously injected into rats, the peak cell concentration in the arterial circulation was an order of magnitude higher than similarly delivered monolayer-grown MSCs (94.8 ± 29.6 vs. 8.2 ± 5.6/10(6) nucleated blood cells, respectively, p<0.05). At 24 h after intravenous injection of the LacZ-labeled bioreactor-grown MSCs, there was a significant threefold decrease in the LacZ-labeled MSCs trapped in the lungs, with a significant increase in the cells reaching the spleen and liver in comparison to their monolayer MSC counterparts. Bioreactor-grown whole marrow cell cultures yielded smaller MSCs with increased capacity to traverse the pulmonary microcirculation compared with traditionally expanded monolayer MSCs. This may significantly improve the capacity and efficiency of these cells to home to injury sites downstream of the lungs.

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.

Ultra-fast bright field and fluorescence imaging of the dynamics of micrometer-sized objects.

High speed imaging has application in a wide area of industry and scientific research. In medical research, high speed imaging has the potential to reveal insight into mechanisms of action of various therapeutic interventions. Examples include ultrasound assisted thrombolysis, drug delivery, and gene therapy. Visual observation of the ultrasound, microbubble, and biological cell interaction may help the understanding of the dynamic behavior of microbubbles and may eventually lead to better design of such delivery systems. We present the development of a high speed bright field and fluorescence imaging system that incorporates external mechanical waves such as ultrasound. Through collaborative design and contract manufacturing, a high speed imaging system has been successfully developed at the University of Pittsburgh Medical Center. We named the system "UPMC Cam," to refer to the integrated imaging system that includes the multi-frame camera and its unique software control, the customized modular microscope, the customized laser delivery system, its auxiliary ultrasound generator, and the combined ultrasound and optical imaging chamber for in vitro and in vivo observations. This system is capable of imaging microscopic bright field and fluorescence movies at 25 × 10(6) frames per second for 128 frames, with a frame size of 920 × 616 pixels. Example images of microbubble under ultrasound are shown to demonstrate the potential application of the system.

Acoustic radiation force for vascular cell therapy: in vitro validation.

Cell-based therapeutic approaches are attractive for the restoration of the protective endothelial layer in arteries affected by atherosclerosis or following angioplasty and stenting. We have recently demonstrated a novel technique for the delivery of mesenchymal stem cells (MSCs) that are surface-coated with cationic lipid microbubbles (MBs) and displaced by acoustic radiation force (ARF) to a site of arterial injury. The objective of this study was to characterize ultrasound parameters for effective acoustic-based delivery of cell therapy. In vitro experiments were performed in a vascular flow phantom where MB-tagged MSCs were delivered toward the phantom wall using ARF generated with an intravascular ultrasound catheter. The translation motion velocity and adhesion of the MB-cell complexes were analyzed. Experimental data indicated that MSC radial velocity and adhesion to the vessel phantom increased with the time-averaged ultrasound intensity up to 1.65 W/cm², after which no further significant adhesion was observed. Temperature increase from baseline near the catheter was 5.5 ± 0.8°C with this setting. Using higher time-averaged ultrasound intensities may not significantly benefit the adhesion of MB-cell complexes to the target vessel wall (p = NS), but could cause undesirable biologic effects such as heating to the MB-cell complexes and surrounding tissue. For the highest time-averaged ultrasound intensity of 6.60 W/cm², the temperature increase was 11.6 ± 1.3°C.

Ultrasound-targeted microbubble destruction to deliver siRNA cancer therapy.

Microbubble contrast agents can specifically deliver nucleic acids to target tissues when exposed to ultrasound treatment parameters that mediate microbubble destruction. In this study, we evaluated whether microbubbles and ultrasound-targeted microbubble destruction (UTMD) could be used to enhance delivery of EGF receptor (EGFR)-directed siRNA to murine squamous cell carcinomas. Custom-designed microbubbles efficiently bound siRNA and mediated RNAse protection. UTMD-mediated delivery of microbubbles loaded with EGFR-directed siRNA to murine squamous carcinoma cells in vitro reduced EGFR expression and EGF-dependent growth, relative to delivery of control siRNA. Similarly, serial UTMD-mediated delivery of EGFR siRNA to squamous cell carcinoma in vivo decreased EGFR expression and increased tumor doubling time, relative to controls receiving EGFR siRNA-loaded microbubbles but not ultrasound or control siRNA-loaded microbubbles and UTMD. Taken together, our results offer a preclinical proof-of-concept for customized microbubbles and UTMD to deliver gene-targeted siRNA for cancer therapy.

In vivo PEG modification of vascular surfaces for targeted delivery.

OBJECTIVE: Thrombosis and restenosis remain problematic for many intravascular procedures. Previously, it has been demonstrated that modifying an injured vascular surface with a protein-reactive polymer could block undesirable platelet deposition. As an added benefit, it would be advantageous if one could target therapeutics to the injured site. This study investigates a site-specific delivery system to target microspheres to vascular surfaces modified with a reactive polyethylene glycol tagged with biotin. METHODS: Rabbit femoral arteries were injured with a 2F embolectomy catheter. Modification of the vascular surface was achieved using a channeled balloon catheter or small-diameter tube. Microspheres were injected intravenously through catheterization of the ear vein. Polymer modification on the injured surface and delivery of microspheres was quantified using epifluorescence microscopy at 0, 24, 48, and 72 hours. RESULTS: Polymer modification of the vascular surface could be achieved using a channeled drug delivery catheter or small-diameter tube with similar results. Maximum polymer coverage occurred at 0 hours and decreased to 85% maximal at 24 hours, 72% at 48 hours, and 67% at 72 hours. The initial number of microspheres per mm(2) binding to modified, injured arteries was 304 versus 141 for the unmodified, damaged control (P < .01). At subsequent times, the number of adherent microspheres to modified, injured arteries decreased by 50%, 70%, and 84% at 24, 48, and 72 hours, respectively; while nonspecific binding to unmodified, injured arteries quickly decreased by 93%. Initial microsphere binding to modified, healthy arteries was 153 microspheres/mm(2) as opposed to 26 microspheres/mm(2) for the unmodified, healthy controls (P < .01). CONCLUSIONS: Chemical modification of injured vessels following intravascular procedures can be readily accomplished in vivo to create a substrate for targeted delivery systems. As a proof of concept, targeted microspheres preferentially adhered to polymer-modified surfaces as opposed to injured, unmodified, or healthy vascular surfaces.

Gene therapy of carcinoma using ultrasound-targeted microbubble destruction.

When microbubble contrast agents are loaded with genes and systemically injected, ultrasound-targeted microbubble destruction (UTMD) facilitates focused delivery of genes to target tissues. A mouse model of squamous cell carcinoma was used to test the hypothesis that UTMD would specifically transduce tumor tissue and slow tumor growth when treated with herpes simplex virus thymidine kinase (TK) and ganciclovir. UTMD-mediated delivery of reporter genes resulted in tumor expression of luciferase and green fluorescent protein (GFP) in perivascular areas and individual tumor cells that exceeded expression in control tumors (p=0.02). The doubling time of TK-treated tumors was longer than GFP-treated tumors (p=0.02), and TK-treated tumors displayed increased apoptosis (p=0.04) and more areas of cellular drop-out (p=0.03). These data indicate that UTMD gene therapy can transduce solid tumors and mediate a therapeutic effect. UTMD is a promising nonviral method for targeting gene therapy that may be useful in a spectrum of tumors.

Vascular endoluminal delivery of mesenchymal stem cells using acoustic radiation force.

Restoration of functional endothelium is a requirement for preventing late stent thrombosis. We propose a novel method for targeted delivery of stem cells to a site of arterial injury using ultrasound-generated acoustic radiation force. Mesenchymal stem cells (MSCs) were surface-coated electrostatically with cationic gas-filled lipid microbubbles (mb-MSC). mb-MSC was characterized microscopically and by flow cytometry. The effect of ultrasound (5 MHz) on directing mb-MSC movement toward the vessel wall under physiologic flow conditions was tested in vitro in a vessel phantom. In vivo testing of acoustic radiation force-mediated delivery of mb-MSCs to balloon-injured aorta was performed in rabbits using intravascular ultrasound (1.7 MHz) during intra-aortic infusion of mb-MSCs. Application of ultrasound led to marginalization and adhesion of mb-MSCs to the vessel phantom wall, whereas no effect was observed on mb-MSCs in the absence of ultrasound. The effect was maximal when there were 7±1 microbubbles/cell (n=6). In rabbits (n=6), adherent MSCs were observed in the ultrasound-treated aortic segment 20 min after the injection (334±137 MSCs/cm(2)), whereas minimal adhesion was observed in control segments not exposed to ultrasound (2±1 MSCs/cm(2), p<0.05). At 24 h after mb-MSC injection and ultrasound treatment, the engrafted MSCs persisted and spread out on the luminal surface of the artery. The data demonstrate proof of principle that acoustic radiation force can target delivery of therapeutic cells to a specific endovascular treatment site. This approach may be used for endoluminal cellular paving and could provide a powerful tool for cell-based re-endothelialization of injured arterial segments.

Optimization of ultrasound contrast agents with computational models to improve selection of ligands and binding strength.

Diagnosis of cardiovascular disease is currently limited by the testing modality. Serum tests for biomarkers can provide quantification of severity but lack the ability to localize the source of the cardiovascular disease, while imaging technology such as angiography and ultrasound can only determine areas of reduced flow but not the severity of tissue ischemia. Targeted imaging with ultrasound contrast agents offers the ability to locally image as well as determine the degree of ischemia by utilizing agents that will cause the contrast agent to home to the affected tissue. Ultrasound molecular imaging via targeted microbubbles (MB) is currently limited by its sensitivity to molecular markers of disease relative to other techniques (e.g., radiolabeling). We hypothesize that computational modeling may provide a useful first approach to maximize microbubble binding by defining key parameters governing adhesion. Adhesive dynamics (AD) was used to simulate the fluid dynamic and stochastic molecular binding of microbubbles to inflamed endothelial cells. Sialyl Lewis(X) (sLe(x)), P-selectin aptamer (PSA), and ICAM-1 antibody (abICAM) were modeled as the targeting receptors on the microbubble surface in both single- and dual-targeted arrangements. Microbubble properties (radius [R(c)], kinetics [k(f), k(r)], and densities of targeting receptors) and the physical environment (shear rate and target ligand densities) were modeled. The kinetics for sLe(x) and PSA were measured with surface plasmon resonance. R(c), shear rate, and densities of sLe(x), PSA, or abICAM were varied independently to assess model sensitivity. Firm adhesion was defined as MB velocity <2% of the free stream velocity. AD simulations revealed an optimal microbubble radius of 1-2 µm and thresholds for kf(in) ( >10(2) s(-1)) and kr(o) (<10(-3) s(-1)) for firm adhesion in a multi-targeted system. State diagrams for multi-targeted microbubbles suggest sLe(x) and abICAM microbubbles may require 10-fold more ligand to achieve firm adhesion at higher shear rates than sLe(x) and PSA microbubbles. The AD model gives useful insight into the key parameters for stable microbubble binding, and may allow flexible, prospective design, and optimization of microbubbles to enhance clinical translation of ultrasound molecular imaging.