Binding Of Microbubbles Research Articles

Ultrasound Molecular Imaging of Neovascularization for Evaluation of Endometrial Receptivity Using Magnetic iRGD-Modified Lipid-Polymer Hybrid Microbubbles.

Angiogenesis plays an important role in endometrial receptivity, determining the response of the endometrium to the blastocyst at the early stage of embryo implantation. During the application of assisted reproduction technologies, it is very important to evaluate the status of uterine angiogenesis before deciding on embryo implantation. Targeted microbubbles (MBs)-based ultrasound molecular imaging (UMI) can noninvasively detect the expression status of biomarkers at the molecular level, thereby being a potential diagnosis strategy for various diseases and their therapeutic evaluation. The iRGD-lipopeptide (DSPE-PEG2000-iRGD) conjugate was prepared with iRGD peptides and DSPE-PEG2000-maleimide through the Michael-type addition reaction. Then, the magnetic iRGD-modified lipid-polymer hybrid MBs (Mag-iLPMs) were prepared with the double-emulsification-solvent-evaporation method. Magnetic targeting of Mag-iLPMs was confirmed under the microscope, followed by a rectangular magnet. Next, the in vitro targeted binding of MBs to murine brain-derived endothelial cells.3 (bEnd.3) and human umbilical vein endothelial cells (HUVEC) were evaluated. The ratio of MBs binding to bEnd.3 and HUVEC at the same field was also compared. For in vivo studies, bolus injections of targeted or control MBs were randomly administrated to non-pregnant or pregnant rats on day 5. Then, the uteri were imaged using a VisualSonics Vevo 2100 ultrasound system (Fujifilm VisualSonics Inc., Ontario, Canada) equipped with a high-frequency transducer. Ultrasonic imaging signals were acquired from Mag-iLPMs, and compared with Mag-LPMs, iLPMs, and LPMs. The Mag-iLPMs showed excellent performance in ultrasound contrast imaging and binding affinity to target cells. Using the magnetic field, 10.5- and 12.47-fold higher binding efficiency to bEnd.3 and HUVEC were achieved compared to non-magnetic iLPMs, respectively. Significantly enhanced UMI signals were also observed in the uteri of rats intravenously injected pregnant rats (6.58-fold higher than rats injected with iLPMs). We provided a powerful ultrasonic molecular functional imaging tool for uterine angiogenesis evaluation before embryonic implantation.

Vancomycin-decorated microbubbles as a theranostic agent for Staphylococcus aureus biofilms

Bacterial biofilms are a huge burden on our healthcare systems worldwide. The lack of specificity in diagnostic and treatment possibilities result in difficult-to-treat and persistent infections. The aim of this in vitro study was to investigate if microbubbles targeted specifically to bacteria in biofilms could be used both for diagnosis as well for sonobactericide treatment and demonstrate their theranostic potential for biofilm infection management. The antibiotic vancomycin was chemically coupled to the lipid shell of microbubbles and validated using mass spectrometry and high-axial resolution 4Pi confocal microscopy. Theranostic proof-of-principle was investigated by demonstrating the specific binding of vancomycin-decorated microbubbles (vMB) to statically and flow grown Staphylococcus aureus (S. aureus) biofilms under increasing shear stress flow conditions (0–12 dyn/cm2), as well as confirmation of microbubble oscillation and biofilm disruption upon ultrasound exposure (2 MHz, 250 kPa, and 5,000 or 10,000 cycles) during flow shear stress of 5 dyn/cm2 using time-lapse confocal microscopy combined with the Brandaris 128 ultra-high-speed camera. Vancomycin was successfully incorporated into the microbubble lipid shell. vMB bound significantly more often than control microbubbles to biofilms, also in the presence of free vancomycin (up to 1000 µg/mL) and remained bound under increasing shear stress flow conditions (up to 12 dyn/cm2). Upon ultrasound insonification biofilm area was reduced of up to 28%, as confirmed by confocal microscopy. Our results confirm the successful production of vMB and support their potential as a new theranostic tool for S. aureus biofilm infections by allowing for specific bacterial detection and biofilm disruption.

Molecular Ultrasound Imaging.

In the last decade, molecular ultrasound imaging has been rapidly progressing. It has proven promising to diagnose angiogenesis, inflammation, and thrombosis, and many intravascular targets, such as VEGFR2, integrins, and selectins, have been successfully visualized in vivo. Furthermore, pre-clinical studies demonstrated that molecular ultrasound increased sensitivity and specificity in disease detection, classification, and therapy response monitoring compared to current clinically applied ultrasound technologies. Several techniques were developed to detect target-bound microbubbles comprising sensitive particle acoustic quantification (SPAQ), destruction-replenishment analysis, and dwelling time assessment. Moreover, some groups tried to assess microbubble binding by a change in their echogenicity after target binding. These techniques can be complemented by radiation force ultrasound improving target binding by pushing microbubbles to vessel walls. Two targeted microbubble formulations are already in clinical trials for tumor detection and liver lesion characterization, and further clinical scale targeted microbubbles are prepared for clinical translation. The recent enormous progress in the field of molecular ultrasound imaging is summarized in this review article by introducing the most relevant detection technologies, concepts for targeted nano- and micro-bubbles, as well as their applications to characterize various diseases. Finally, progress in clinical translation is highlighted, and roadblocks are discussed that currently slow the clinical translation.

Pharmacokinetic Modeling of Targeted Ultrasound Contrast Agents for Quantitative Assessment of Anti-Angiogenic Therapy: a Longitudinal Case-Control Study in Colon Cancer

PurposeTo evaluate quantitative and semi-quantitative ultrasound molecular imaging (USMI) for antiangiogenic therapy monitoring in human colon cancer xenografts in mice.ProceduresColon cancer was established in 17 mice by injection of LS174T (Nr = 9) or CT26 (Nn = 8) cancer cells to simulate clinical responders and non-responders, respectively. Antiangiogenic treatment (bevacizumab; Nrt = Nnt = 5) or control treatment (saline; Nrc = 4, Nnc = 3) was administered at days 0, 3, and 7. Three-dimensional USMI was performed by injection at days 0, 1, 3, 7, and 10 of microbubbles targeted to the vascular endothelial growth factor receptor 2 (VEGFR2). Microbubble binding rate (kb), estimated by first-pass binding model fitting, and semi-quantitative parameters late enhancement (LE) and differential targeted enhancement (dTE) were compared at each day to evaluate their ability to assess and predict the response to therapy. Correlation analysis with the ex-vivo immunohistological quantification of VEGFR2 expression and the percentage blood vessel area was also performed.ResultsSignificant changes in the USMI parameters during treatment were observed only in the responders treated with bevacizumab (p-value < 0.05). Prediction of the response to therapy as early as 1 day after treatment was achieved by the quantitative parameter kb (p-value < 0.01), earlier than possible by tumor volume quantification. USMI parameters could significantly distinguish between clinical responders and non-responders (p-value << 0.01) and correlated well with the ex-vivo quantification of VEGFR2 expression and the percentage blood vessels area (p-value << 0.01).ConclusionUSMI (semi)quantitative parameters provide earlier assessment of the response to therapy compared to tumor volume, permit early prediction of non-responders, and correlate well with ex-vivo angiogenesis biomarkers.

Abstract P2-04-04: Developing targeted therapeutic microbubbles for enhanced epigenetic drug delivery for breast cancer

Abstract Background: In recent years, there has been great interest in developing drugs that modify epigenetic changes as new therapies for breast cancer (BC). There is evidence that aberrant epigenetic inactivation of genes, essential for normal cell growth, is involved in cancer. These modifications are potentially reversible therefore re-activation of genes in response to epigenetic drugs can result in inhibition of tumour growth or sensitisation to other anti-cancer therapies. Epigenetic drugs are in clinical trials for BC but have some drawbacks: while some can be highly effective in vitro, their poor stability could compromise their clinical use. Also, high doses required to induce an effect in patients could increase off-site toxicity. As a result there is an urgent need to develop novel systems for the delivery and release of these drugs. Objectives: Our aim is to develop targeted microbubbles (MBs) to enhance therapeutic effects in vitro and in vivo. Ultrasound (US)-mediated drug delivery using MBs is proposed as a non-invasive approach for localised drug administration. Methods: We developed assays using low doses of a DNA methyltransferase inhibitor, called decitabine (DAC), for determining its delivery and effect in vitro and in vivo. VEGFR2 was assessed as a targeting molecule for therapeutic delivery to tumour vasculature in vitro and in a human BC xenograft model. We have generated therapeutic MBs with DAC using a flow-focussing microfluidic platform and conducted in vitro and currently performing in vivo studies to observe tumour and tissue responses. Results: Treatment of triple-negative BC (TNBC) cells with low DAC doses revealed restoration of epigenetically dysregulated tumour suppressor genes. These genes were used as biomarkers for the assessment of DAC effects in a human TNBC mouse model. To evaluate the use of a targeting molecule for drug delivery, specific binding of VEGFR2-targeted MBs on VEGFR2+ve mouse endothelial cells was verified by a flow assay. VEGFR2 expression was assessed longitudinally in xenograft tissue and demonstrated significantly higher VEGFR2 expression in the vasculature of smaller size tumours, indicating the time that delivery of targeted MBs would be most effective. DAC-loaded liposomes or co-administration of DAC and MBs in combination with US using a specifically designed US transducer, were tested in vitro. Currently, investigation of the potential of US-triggered drug delivery enhancing efficacy of DAC in vivo is being carried out. In addition, combination treatments have been performed in vitro, showing increased sensitisation of cells to anthracycline treatment after priming with DAC. Conclusions: It may be feasible to combine US, MBs and epigenetic therapy in a pre-clinical setting to improve drug efficacy, particularly for drugs that are rapidly degraded within the body. MB delivery may have the potential to reduce the dosage required, thus reducing off-site side effects in patients. Citation Format: Alataki A, Abou-Saleh R, McLaughlan JR, Markham AF, Evans SD, Coletta PL, Valleley EM. Developing targeted therapeutic microbubbles for enhanced epigenetic drug delivery for breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-04-04.

A molecular intravascular ultrasound contrast agent allows detection of activated platelets on the surface of symptomatic human plaques

Background and aimsActivated platelets are amongst the most attractive imaging targets in atherosclerosis due to their important role in early processes of atherogenesis and thrombus formation. We developed a molecular intravascular ultrasound (IVUS) approach to detect activated platelets ex vivo on the surface of human plaques, using an IVUS system applied in clinical routine. MethodsHuman carotid endarterectomy specimens were obtained directly from the operating room and exposed to artificial arterial flow conditions for incubation with the contrast agent. This consists of microbubbles (MB), which are linked to an antibody against the ligand induced binding site (LIBS) of the activated platelet glycoprotein IIb/IIa, and a sialyl Lewis polymer (SL), which mediates binding to selectins (LIBS-SL-MB). IVUS was performed pre and post incubation with LIBS-SL-MB and after rinsing with PBS. In comparison, IVUS was performed pre and post incubation with MBs linked to an unspecific control antibody and a dysfunctional polymer (control-MB). All imaging results were correlated to histology findings. ResultsIVUS imaging showed a high signal enhancement after administration of LIBS-SL-MB. After rinsing with PBS, the signal enhancement remained stable. Immunofluorescence and immunohistochemistry confirmed significant binding of microbubbles to thrombi on the plaque surface. Moreover, thrombus size and number of bound MBs correlated well. ConclusionsLIBS-SL-MB allows ex vivo IVUS imaging of even small numbers of activated platelets on the surface of human carotid endarterectomy specimens. This diagnostic approach could deliver valuable additional information for risk stratification of atherosclerotic plaques, especially since we apply a clinically well-established IVUS imaging system.

Development of prostate specific membrane antigen targeted ultrasound microbubbles using bioorthogonal chemistry

Prostate specific membrane antigen (PSMA) targeted microbubbles (MBs) were developed using bioorthogonal chemistry. Streptavidin-labeled MBs were treated with a biotinylated tetrazine (MBTz) and targeted to PSMA expressing cells using trans-cyclooctene (TCO)-functionalized anti-PSMA antibodies (TCO-anti-PSMA). The extent of MB binding to PSMA positive cells for two different targeting strategies was determined using an in vitro flow chamber. The initial approach involved pretargeting, where TCO-anti-PSMA was first incubated with PSMA expressing cells and followed by MBTz, which subsequently showed a 2.8 fold increase in the number of bound MBs compared to experiments performed in the absence of TCO-anti-PSMA. Using direct targeting, where TCO-anti-PSMA was linked to MBTz prior to initiation of the assay, a 5-fold increase in binding compared to controls was observed. The direct targeting approach was subsequently evaluated in vivo using a human xenograft tumor model and two different PSMA-targeting antibodies. The US signal enhancements observed were 1.6- and 5.9-fold greater than that for non-targeted MBs. The lead construct was also evaluated in a head-to-head study using mice bearing both PSMA positive or negative tumors in separate limbs. The human PSMA expressing tumors exhibited a 2-fold higher US signal compared to those tumors deficient in human PSMA. The results demonstrate both the feasibility of preparing PSMA-targeted MBs and the benefits of using bioorthogonal chemistry to create targeted US probes.

Microbubble Enzyme-Linked Immunosorbent Assay for the Detection of Targeted Microbubbles in in Vitro Static Binding Assays

Targeted microbubbles (MBs) are ultrasound contrast agents that are functionalized with a ligand for ultrasound molecular imaging of endothelial markers. Novel targeted MBs are characterized in vitro by incubation in protein-coated wells, followed by binding quantification by microscopy or ultrasound imaging. Both methods provide operator-dependent results: Between 3 and 20 fields of view from a heterogeneous sample are typically selected for analysis by microscopy, and in ultrasound imaging, different acoustic settings affect signal intensities. This study proposes a new method to reproducibly quantify MB binding based on enzyme-linked immunosorbent assay (ELISA), in which bound MBs are revealed with an enzyme-linked antibody. MB-ELISA was adapted to in vitro static binding assays, incubating the MBs in inverted position or by agitation, and compared with microscopy. The specificity and sensitivity of MB-ELISA enable the reliable quantification of MB binding in a rapid, high-throughput and whole-well analysis, facilitating the characterization of new targeted contrast agents.

Quantitative ultrasound molecular imaging by modeling the binding kinetics of targeted contrast agent

Ultrasound molecular imaging (USMI) is an emerging technique to monitor diseases at the molecular level by the use of novel targeted ultrasound contrast agents (tUCA). These consist of microbubbles functionalized with targeting ligands with high-affinity for molecular markers of specific disease processes, such as cancer-related angiogenesis. Among the molecular markers of angiogenesis, the vascular endothelial growth factor receptor 2 (VEGFR2) is recognized to play a major role. In response, the clinical-grade tUCA BR55 was recently developed, consisting of VEGFR2-targeting microbubbles which can flow through the entire circulation and accumulate where VEGFR2 is over-expressed, thus causing selective enhancement in areas of active angiogenesis. Discrimination between bound and free microbubbles is crucial to assess cancer angiogenesis. Currently, this is done non-quantitatively by looking at the late enhancement, about 10 min after injection, or by calculation of the differential targeted enhancement, requiring the application of a high-pressure ultrasound (US) burst to destroy all the microbubbles in the acoustic field and isolate the signal coming only from bound microbubbles. In this work, we propose a novel method based on mathematical modeling of the binding kinetics during the tUCA first pass, thus reducing the acquisition time and with no need for a destructive US burst. Fitting time-intensity curves measured with USMI by the proposed model enables the assessment of cancer angiogenesis at both the vascular and molecular levels. This is achieved by estimation of quantitative parameters related to the microvascular architecture and microbubble binding. The proposed method was tested in 11 prostate-tumor bearing rats by performing USMI after injection of BR55, and showed good agreement with current USMI methods. The novel information provided by the proposed method, possibly combined with the current non-quantitative methods, may bring deeper insight into cancer angiogenesis, and thus potentially improve cancer diagnosis and management.

Development of a new therapeutic technique to direct stem cells to the infarcted heart using targeted microbubbles: StemBells

Successful stem cell therapy after acute myocardial infarction (AMI) is hindered by lack of engraftment of sufficient stem cells at the site of injury. We designed a novel technique to overcome this problem by assembling stem cell-microbubble complexes, named ‘StemBells’.StemBells were assembled through binding of dual-targeted microbubbles (~3μm) to adipose-derived stem cells (ASCs) via a CD90 antibody. StemBells were targeted to the infarct area via an ICAM-1 antibody on the microbubbles. StemBells were characterized microscopically and by flow cytometry. The effect of ultrasound on directing StemBells towards the vessel wall was demonstrated in an in vitro flow model. In a rat AMI-reperfusion model, StemBells or ASCs were injected one week post-infarction. A pilot study demonstrated feasibility of intravenous StemBell injection, resulting in localization in ICAM-1-positive infarct area three hours post-injection. In a functional study five weeks after injection of StemBells cardiac function was significantly improved compared with controls, as monitored by 2D-echocardiography. This functional improvement neither coincided with a reduction in infarct size as determined by histochemical analysis, nor with a change in anti- and pro-inflammatory macrophages.In conclusion, the StemBell technique is a novel and feasible method, able to improve cardiac function post-AMI in rats.

Low-Dose Molecular Ultrasound Imaging with E-Selectin-Targeted PBCA Microbubbles.

Our objective was to determine the lowest diagnostically effective dose for E-selectin-targeted poly n-butyl cyanoacrylate (PBCA)-shelled microbubbles and to apply it to monitor antiangiogenic therapy effects. PBCA-shelled microbubbles (MBs) coupled to an E-selectin-specific peptide were applied in mice carrying MLS or A431 carcinoma xenografts scaling down the MB dosage to the lowest level where binding could be examined with a 18-MHz small animal ultrasound transducer. Differences in E-selectin expression in the two carcinoma xenografts were confirmed by enzyme-linked immunosorbent assay (ELISA). In addition, MLS tumor-bearing mice under antiangiogenic therapy were monitored using E-selectin-targeted MBs at the lowest applicable dose. Therapy effects on tumor vascularization were verified by immunohistological analyses. The minimally required dosage was 7 × 10(7) MBs/kg body weight. This dosage was sufficient to enable E-selectin detection in high E-selectin-expressing MLS tumors, while low E-selectin-expressing A431 tumors required almost 2.5-fold higher doses. At the dose of 7 × 10(7) MBs/kg body weight, a decrease in E-selectin MB binding under antiangiogenic therapy could be assessed (being significant after 3 days of treatment; p < 0.0001), which was in line with the significant drop in E-selectin-positive area fractions that was found histologically (p < 0.05). Molecular ultrasound imaging with our E-selectin-targeted MB and therapy monitoring was possible down to a dose of 7 × 10(7) MBs/kg body weight (equates to 66 μg PBCA/kg and 4.6 mg PBCA/70 kg). Improvements in choice of targets, MB composition, and other MB detection methods may improve sensitivity and lead to reliable detection results of clinically transferrable MBs at even lower dosage levels.

Molecular imaging using ultrasound and microbubbles

Increasing insight into the molecular mechanisms of disease has created a need to visualize sub-cellular events in clinical populations in order for treatment paradigms to advance. Molecular imaging has emerged as an important translational tool for understanding disease pathogenesis, for which various probes and detection methods have been developed. Ultrasonic detection of molecular markers of disease using acoustically active particles is one such in vivo modality. Molecular imaging with ultrasound utilizes intravascular delivery of gas-filled microspheres (microbubbles) targeted to bind to disease-specific epitopes expressed by vascular endothelial cells. Microbubble binding occurs by virtue of targeting ligands attached to the microbubble surface, which confer specific binding to the endothelial target. In the presence of ultrasound, the bound microbubbles emit an acoustic signal which can be detected as a persistent contrast effect during ultrasound imaging with non-linear based detection strategies. In pre-clinical models, ultrasound molecular imaging has been used to detect early atherosclerotic disease, myocardial ischemic memory (recent myocardial ischemia), acute heart transplant rejection, angiogenesis, and for in vivo tracking of therapeutically administered stem cells. Such applications raise exciting possibilities for meaningful bedside translation of this imaging platform to address important clinical issues in an era of molecular medicine.

Contrast Imaging in Mouse Embryos Using High-frequency Ultrasound

Ultrasound contrast-enhanced imaging can convey essential quantitative information regarding tissue vascularity and perfusion and, in targeted applications, facilitate the detection and measure of vascular biomarkers at the molecular level. Within the mouse embryo, this noninvasive technique may be used to uncover basic mechanisms underlying vascular development in the early mouse circulatory system and in genetic models of cardiovascular disease. The mouse embryo also presents as an excellent model for studying the adhesion of microbubbles to angiogenic targets (including vascular endothelial growth factor receptor 2 (VEGFR2) or αvβ3) and for assessing the quantitative nature of molecular ultrasound. We therefore developed a method to introduce ultrasound contrast agents into the vasculature of living, isolated embryos. This allows freedom in terms of injection control and positioning, reproducibility of the imaging plane without obstruction and motion, and simplified image analysis and quantification. Late gestational stage (embryonic day (E)16.6 and E17.5) murine embryos were isolated from the uterus, gently exteriorized from the yolk sac and microbubble contrast agents were injected into veins accessible on the chorionic surface of the placental disc. Nonlinear contrast ultrasound imaging was then employed to collect a number of basic perfusion parameters (peak enhancement, wash-in rate and time to peak) and quantify targeted microbubble binding in an endoglin mouse model. We show the successful circulation of microbubbles within living embryos and the utility of this approach in characterizing embryonic vasculature and microbubble behavior.

Contrast imaging in mouse embryos using high-frequency ultrasound.

Ultrasound contrast-enhanced imaging can convey essential quantitative information regarding tissue vascularity and perfusion and, in targeted applications, facilitate the detection and measure of vascular biomarkers at the molecular level. Within the mouse embryo, this noninvasive technique may be used to uncover basic mechanisms underlying vascular development in the early mouse circulatory system and in genetic models of cardiovascular disease. The mouse embryo also presents as an excellent model for studying the adhesion of microbubbles to angiogenic targets (including vascular endothelial growth factor receptor 2 (VEGFR2) or αvβ3) and for assessing the quantitative nature of molecular ultrasound. We therefore developed a method to introduce ultrasound contrast agents into the vasculature of living, isolated embryos. This allows freedom in terms of injection control and positioning, reproducibility of the imaging plane without obstruction and motion, and simplified image analysis and quantification. Late gestational stage (embryonic day (E)16.6 and E17.5) murine embryos were isolated from the uterus, gently exteriorized from the yolk sac and microbubble contrast agents were injected into veins accessible on the chorionic surface of the placental disc. Nonlinear contrast ultrasound imaging was then employed to collect a number of basic perfusion parameters (peak enhancement, wash-in rate and time to peak) and quantify targeted microbubble binding in an endoglin mouse model. We show the successful circulation of microbubbles within living embryos and the utility of this approach in characterizing embryonic vasculature and microbubble behavior.

Contrast-enhanced molecular ultrasound differentiates endoglin genotypes in mouse embryos

Targeted ultrasound contrast imaging has the potential to become a reliable molecular imaging tool. A better understanding of the quantitative aspects of molecular ultrasound technology could facilitate the translation of this technique to the clinic for the purposes of assessing vascular pathology and detecting individual response to treatment. The objective of this study was to evaluate whether targeted ultrasound contrast-enhanced imaging can provide a quantitative measure of endogenous biomarkers. Endoglin, an endothelial biomarker involved in the processes of development, vascular homeostasis, and altered in diseases, including hereditary hemorrhagic telangiectasia type 1 and tumor angiogenesis, was the selected target. We used a parallel plate perfusion chamber in which endoglin-targeted (MBE), rat isotype IgG2 control and untargeted microbubbles were perfused across endoglin wild-type (Eng+/+), heterozygous (Eng+/-) and null (Eng-/-) embryonic mouse endothelial cells and their adhesion quantified. Microbubble binding was also assessed in late-gestation, isolated living transgenic Eng+/- and Eng+/+ embryos. Nonlinear contrast-specific ultrasound imaging performed at 21 MHz was used to collect contrast mean power ratios for all bubble types. Statistically significant differences in microbubble binding were found across genotypes for both in vitro (p<0.05) and embryonic studies (p<0.001); MBE binding was approximately twofold higher in Eng+/+ cells and embryos compared with their Eng+/- counterparts. These results suggest that molecular ultrasound is capable of reliably differentiating between molecular genotypes and relating receptor densities to quantifiable molecular ultrasound levels.

Abstract 2060: Quantifying vascular biomarkers with contrast-enhanced molecular ultrasound imaging

Abstract Introduction: Molecular imaging has the potential to dramatically impact all facets of patient care, from early disease detection to treatment monitoring and follow-up, as a tool for the characterization and measurement of key biomolecules in vivo. In ultrasound, functional and molecular imaging is possible through the use of microbubbles (MB), a contrast agent that can be transformed into targeting agents that bind to vascular biomarkers of interest. In this study, we evaluate whether targeted ultrasound contrast enhanced imaging can provide a quantitative measure of surface receptor expression in endothelial cell populations. Methods: The biomarker endoglin (Eng) was selected for targeting due to its involvement in the processes of development, vascular regulation and disease, including tumor angiogenesis. Endoglin wildtype (Eng+/+), heterozygous null (Eng+/-) and null (Eng-/-) mouse embryonic endothelial cells were cultured on glass slides and mounted in parallel plate flow chambers. MicroMarker microbubbles (endoglin targeted: MBE, isotype control: MBC or untargeted: MBU at 1x107 MB/mL in PBS) were perfused across the cells at 4 mL/min, corresponding to a shear stress of 2 dynes/cm2. Cell and bubble numbers were determined from bright field and phase images (Nikon, 40x), with adhesion quantified as the number of MB/cell. Binding of microbubbles was also assessed in late-gestational stage, isolated, living embryos (Eng+/+, Eng+/-). The highly regulated and controlled activities of normal angiogenesis and vasculogenesis in the mouse embryo make it an excellent surrogate for complex and heterogeneous tumor microenvironments, while genetic manipulation enables the generation of a variety of useful transgenic models. Nonlinear contrast-specific ultrasound imaging, performed at 21MHz with a Vevo-2100 scanner (VisualSonics Inc.), was used to collect contrast mean power ratios (CMPR, representative measure of MB binding) within the brains of each embryo 4 minutes after a bolus injection of MBE, MBC or MBU. Results: Expression levels in cells and embryos were significantly different across genotypes, with endoglin reduced by half in Eng+/- and totally absent in Eng-/- samples. In vitro, microbubble adhesion was found to vary significantly (p&amp;lt;0.05) across genotype populations, with minimal attachment of MBC and MBU compared to MBE. Endoglin-targeted binding was approximately two-fold higher (median = 0.96 MBE/cell) in Eng+/+ compared to Eng+/- (median = 0.42 MBE/cell) cells. In embryo studies, we observed minimal signal from MBC and MBU, while MBE binding was found to be significantly higher in Eng+/+ embryos (CMPR+/+ = 9.71 + 0.66, 95% CI) compared to Eng+/- embryos (CMPR+/- = 5.51 + 0.64, 95% CI). In conclusion, these results suggest that molecular ultrasound is capable of reliably differentiating between molecular genotypes and relating receptor densities to quantifiable molecular ultrasound levels. Citation Format: Janet M. Denbeigh, Brian A. Nixon, John J.Y. Lee, Mirjana Jerkic, Philip A. Marsden, Michelle Letarte, Mira C. Puri, F. Stuart Foster. Quantifying vascular biomarkers with contrast-enhanced molecular ultrasound imaging. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2060. doi:10.1158/1538-7445.AM2014-2060

Dynamics of Targeted Microbubble Adhesion Under Pulsatile Compared with Steady Flow

Hemodynamic flow variations at low fluid shear stress are thought to play a critical role in local atherosclerotic plaque initiation and development and to affect plaque instability. Targeted microbubbles are being developed as intravascular agents for identifying atherosclerotic lesions using ultrasound. How variations in local hydrodynamic flow influence the adhesiveness of targeted microbubbles is not well understood. We postulated that rates of targeted microbubble binding and accumulation differ when subjected to steady flow (SF) as compared with oscillatory or pulsatile flow (PF), because PF imposes non-uniform blood rheology and periodic acceleration and deceleration of blood velocity, when compared with SF. We assessed the binding rates of targeted microbubbles in seven randomly assigned PF and seven matched SF replicate runs at low (<1 Pa) and intermediate (≥1 and <2.5 Pa) wall shear stress (WSS) by drawing 4.8 × 106 microbubbles mL−1 over streptavidin-coated substrates, immobilized within a parallel plate flow chamber at a calculated density of 81 binding sites μm-2. Selective binding and accumulation of targeted microbubbles was recorded in a single field of view using real-time video microscopy. Microbubble accumulation was modeled to obtain flow-mediated microbubble binding kinetics (amplitude, A, and rate constant, k). PF elicited higher microbubble accumulation rates, in comparison to SF. The rates of microbubble accumulation differed significantly between PF and SF (p < 0.05) at intermediate WSS but not at low WSS (p > 0.05). The rate of microbubble accumulation decreased as WSS increased.

VEGFR2-Targeted Molecular Imaging in the Mouse Embryo: An Alternative to the Tumor Model

As a tumor surrogate, the mouse embryo presents as an excellent alternative for examining the binding of angiogenesis-targeting microbubbles and assessing the quantitative nature of molecular ultrasound. We establish the validity of this model by developing a robust method to study microbubble kinetic behavior and investigate the reproducibility of targeted binding in the murine embryo. Vascular endothelial growth factor receptor 2 (VEGFR2)-targeted (MBV), rat immunoglobulin G2 (IgG2) control antibody-targeted (MBC) and untargeted (MBU) microbubbles were introduced into vasculature of living mouse embryos. Non-linear contrast-specific and B-mode ultrasound imaging, performed at 21 MHz with a Vevo-2100 scanner, was used to collect basic perfusion parameters and contrast mean power ratios for all bubble types. We observed a twofold increase (p < 0.001) in contrast mean power ratios for MBV (4.14 ± 1.78) compared with those for MBC (1.95 ± 0.78) and MBU (1.79 ± 0.45). Targeted imaging of endogenous endothelial cell surface markers in mouse embryos is possible with labeled microbubbles. The mouse embryo thus presents as a versatile model for testing the performance of ultrasound molecular targeting, where further development of quantitative imaging techniques may enable rapid evaluations of biomarker expression in studies of vascular development, disease and angiogenesis.

Binding and isolation of tumor cells in biological media with perfluorocarbon microbubbles

With the emerging interest in personalized medicine, there is strong demand for new technologies for clinical sample interrogation. Exfoliated tumor cells in variety of pathological samples (e.g., blood, bone marrow, urine) could provide invaluable information for diagnosis and prognosis of cancers. Here we describe a detailed method for capture and isolation of tumor cells in medium, blood, or large issue buffy coat using EpCAM-targeted buoyant microbubbles (MBs). Perflorohexane gas lipid shell MBs were prepared with emulsification method and conjugated with antibody as described by us before [25]. The binding of EpCAM-targeted MBs to A549 (human lung carcinoma) and 4T1 (mouse breast carcinoma) cells spiked into BSA/PBS or blood was more than 90%, which was comparable with commercial anti-EpCAM immunomagnetic beads (DynaBeads). Anti-EpCAM MBs efficiently (75–82%) isolated BxPC3 pancreatic tumor cells spiked into medium, blood or a buffy coat, within 15–30min of incubation. We discuss MB parameters and experimental conditions critical to achieve efficient cells binding and isolation. In conclusion, MB-assisted cell isolation is a promising method for rapid enrichment of cells and biomarkers from biological samples.

Directing stem cells to the infarcted heart using targeted microbubbles: development of a new molecular therapeutic technique

Stem cell therapy is a promising tool to restore contractile function after myocardial infarction. Unfortunately, clinical trials still show disappointing results with only minor improvements in cardiac function. The major problem of cellular therapy is lack of persistence of sufficient stem cells at the site of injury, independent of administration route. We designed a novel technique to overcome this problem by directing stem cells to the infarcted area using targeted microbubbles (MB) and ultrasound (US). For this we coupled adipose-derived stem cells (ASC) to MB using an antibody against CD90 via biotin-avidin bridging. This stem cell-bubble complex was named StemBell. StemBells were targeted to the infarcted area via a second antibody on the MB: anti-CD54. US (1 MHz) was applied to exert acoustic radiation force on the StemBells. In vitro we demonstrated that the procedure to create StemBells, as well as exposure to US had no negative effect on cell viability, using flow cytometry. Binding of MB to did not affect their ability to attach to a culture dish, demonstrated by light microscopy. In a flow system we showed the ability of US to push StemBells from the main flow (0.2 dyne/cm2) to the side. In vivo, acute myocardial infarction was mimicked in a rat by ligating the left anterior descending coronary artery for 40 min, followed by reperfusion. 7 Days post-infarction 1 million DAPI-labeled StemBells (StB) or were injected intravenously. 3 Hours post-injection hearts were excised, stored in liquid N2 and cryo-sections were made. By performing fluorescence microscopy we found significantly more cells (6-fold increase) specifically in the infarcted area in the StB group compared with ASC alone group (n=6; p<0.01). Applying US (StB+US) lead to an 8-fold increase (n=6; p<0.01 vs ASC alone, p<0.05 vs StB). Notably, retrieved stem cells coincided with CD54 positive areas. In conclusion, we successfully demonstrated proof-of-principle of a novel technique to increase the number of stem cells at the site of injury. This holds great promise for stem cell therapies in general.