Ultrasound Molecular Imaging for the Guidance of Ultrasound-Triggered Release of Liposomal Doxorubicin and Its Treatment Monitoring in an Orthotopic Prostatic Tumor Model in Rat.

Liposome encapsulation of drugs is an interesting approach in cancer therapy to specifically release the encapsulated drug at the desired treatment site. In addition to thermo-, pH-, light-, enzyme- or redox-responsive liposomes, which have had promising results in (pre-) clinical studies, ultrasound-triggered sonosensitive liposomes represent an exciting alternative to locally trigger the release from these cargos. Localized drug release requires precise tumor visualization to produce a targeted and ultrasound stimulus. We used ultrasound molecular imaging (USMI) with BR55, a vascular endothelial growth factor receptor 2 (VEGFR2)-targeted ultrasound contrast agent, to guide ultrasound-triggered release of sonosensitive liposomes encapsulating doxorubicin (L-DXR) in an orthotopic prostatic rodent tumor model. Forty-eight hours after L-DXR injection, local release of doxorubicin was triggered with a confocal ultrasound device with two focused transducers, 1.1-MHz center frequency, and peak positive and negative pressures of 20.5 and 13 MPa at focus. Tumor size decreased by 20% in 2 wk with L-DXR alone (n = 9) and by 70% after treatment with L-DXR and confocal ultrasound (n = 7) (p < 0.01). The effect of doxorubicin on perfusion/vascularity and VEGFR2 expression was evaluated by USMI and immunohistochemistry of CD31 and VEGFR2 and did not reveal differences in perfusion or VEGFR2 expression in the absence or after the triggered release of liposomes. USMI can provide precise guidance for ultrasound-triggered release of liposomal doxorubicin mediated by a confocal ultrasound device; moreover, the combination of B-mode imaging and USMI can help to follow the response of the tumor to the therapy.

Ultrasound Molecular Imaging With BR55, a Predictive Tool of Antiangiogenic Treatment Efficacy in a Chemo-Induced Mammary Tumor Model.

OBJECTIVES: The aim of this study was to evaluate the added value of ultrasound molecular imaging of the vascular growth factor receptor 2 (VEGFR2) expression, using the clinical grade contrast agent BR55, for the early evaluation of antiangiogenic treatment efficacy in a chemo-induced rat mammary tumor model. MATERIALS AND METHODS: In this preclinical study, chemo-induced rat mammary tumors were obtained after a single injection of N-nitroso-N-methylurea intraperitoneally in 46 prepubescent (age 38 ± 2 days) female rats. All experiments were performed under the authorization of the Direction Générale de la Santé, Geneva, Switzerland. Once tumor reached 0.8 cm in the largest cross-section, animals were enrolled in a sunitinib- or vehicle-treated group. Ultrasound molecular imaging was performed using BR55, a clinical grade targeted contrast agent against VEGFR2, before therapy and up to 72 hours. Anatomical changes of tumor over time, that is, area of the tumor largest cross-section and tumor volume, were measured in B-mode. Signal from microbubbles was detected in a nonlinear contrast mode (power modulation) using the iU22 diagnostic ultrasound system (Phillips, United States) equipped with a L12-5 linear transducer (transmit frequency 5 MHz). Peak enhancement and wash-in area under the curve were extracted from the time intensity curves generated by a dedicated quantification software for contrast ultrasound, so-called VueBox (Bracco Suisse SA, Switzerland). The signal of bound BR55 microbubbles in the tumor was quantified 10 minutes after injection. Altogether, these parameters were used to monitor tumoral response to treatment at the anatomical, functional, and molecular levels. At each time point, a cohort of tumors was harvested for the assessment of CD31 and VEGFR2 expression by immunohistochemistry staining. RESULTS: Under sunitinib therapy, assessment of the expression of VEGFR2 by ultrasound molecular imaging with BR55 reveals a significant difference as early as 12 hours after first dosing (-25%), whereas tumor size significant change occurs only after 24 hours. At the end of the therapeutic protocol, 72 hours after the onset of treatment, molecular changes are more marked with a 80% decrease compared with only ~40% for the anatomic parameters. Ultrasound molecular imaging observations suggesting a decrease in VEGFR2 expression in treated tumors were corroborated by semiquantitative grading of VEGFR2, showing a decrease expression over time. Functional parameters measured in the perfusion phase also show a decrease along treatment, significant for 24 hours and of 48% of peak enhancement at the end of protocol. CONCLUSIONS: Anatomical, functional, and molecular evaluations are feasible in a single examination using BR55 ultrasound targeted contrast agent. Ultrasound molecular imaging of VEGFR2 can depict an early response to antiangiogenic treatment in a rat mammary tumor model. This imaging modality has a potential for early assessment of each patient's response, which could be useful to take decisions on therapeutic protocol, providing as such an imaging tool for personalized medicine.

Ultrasound molecular imaging of transient acute myocardial ischemia with a clinically translatable P- and E-selectin targeted contrast agent: correlation with the expression of selectins.

OBJECTIVE: The diagnosis of acute coronary syndrome remains challenging especially in patients without clear symptoms or electrocardiographic and/or biomarker features. A hallmark of ischemia/reperfusion is activation of endothelial cells leading to altered expression of molecular markers, including selectins. In this context, we aimed to validate the value of ultrasound molecular imaging for detecting transient myocardial ischemia by using a clinically translatable dual P- and E-selectin-targeted ultrasound contrast agent (UCA) and microbubble (MB(selectin)). MATERIAL AND METHODS: Transient (20 minutes) myocardial ischemia of rat heart was produced by ligation of the left anterior descending coronary artery ligation followed by 2-, 5-, or 24-hour reperfusion. Imaging of the transient ischemic event was achieved by the use of MB(selectin). Performance of this clinically translatable targeted UCA was compared with that of antibody-targeted streptavidin MBs. Finally, immunohistochemistry staining of rat myocardial ischemic tissue was performed to assess expression of selectins accessible to targeted UCA. RESULTS: In rats subjected to myocardial ischemia (20 minutes) followed by reperfusion (2 hours), injection of MB(selectin) produced high late phase (ie, 10-minute postinjection) ultrasound molecular imaging enhancement in the myocardium, which colocalized with the ischemic area. Late phase enhancement persisted 5 and 24 hours after reperfusion. Similarly, the use of MBP and MBE, comprising antibodies specific for P- and E-selectin, respectively, showed high late-phase enhancement within the ischemic area compared with remote myocardial tissue. Two and 5 hours after ischemia has resolved, a persistent expression of these 2 selectins was detected. After 24 hours of reperfusion, only MBE produced late phase enhancement within the ischemic myocardium. Immunohistochemical findings revealed that both P- and E-selectin were expressed and accessible on the surface of the activated endothelium 2 and 5 hours after the acute ischemic event, whereas only E-selectin remained accessible after 24 hours. CONCLUSIONS: Ultrasound molecular imaging of transient myocardial ischemia using dual selectin-targeted UCA is able to monitor the time course of expression of selectins after resolution of the ischemic event, paving the way for a large clinical diagnostic window.

BR55: a lipopeptide-based VEGFR2-targeted ultrasound contrast agent for molecular imaging of angiogenesis.

OBJECTIVES: BR55, an ultrasound contrast agent functionalized with a heterodimer peptide targeting the vascular endothelial growth factor receptor 2 (VEGFR2), was evaluated in vitro and in vivo, demonstrating its potential for specific tumor detection. MATERIALS AND METHODS: The targeted contrast agent was prepared by incorporation of a biospecific lipopeptide into the microbubble membrane. Experiments were performed in vitro to demonstrate the binding capacities of BR55 microbubbles on immobilized receptor proteins and on various endothelial or transfected cells expressing VEGFR2. The performance of BR55 microbubbles was compared with that of streptavidin-conjugated microbubbles targeted to the same receptor by coupling them to a biotinylated antibody. The specificity of BR55 binding to human and mouse endothelial cells was determined in competition experiments with the free lipopeptide, vascular endothelial growth factor (VEGF), or a VEGFR2-specific antibody. Molecular ultrasound imaging of VEGFR2 was performed in an orthotopic breast tumor model in rats using a nondestructive, contrast-specific imaging mode. RESULTS: BR55 was shown to bind specifically to the immobilized recombinant VEGFR2 under flow (dynamic conditions). BR55 accumulation on the target over time was similar to that of microbubbles bearing a specific antibody. BR55 avidly bound to cells expressing VEGFR2, and the pattern of microbubble distribution was correlated with the pattern of receptor expression determined by immunocytochemistry. The binding of targeted microbubbles on cells was competed off by an excess of free lipopeptide, the natural ligand (VEGF) and by a VEGFR2-specific antibody (P < 0.001). Although selected for the human receptor, the VEGFR2-binding lipopeptide was also shown to recognize the rodent receptor. Tumor perfusion was assessed during the vascular phase of BR55, and then the malignant lesion was highlighted by specific accumulation of the targeted microbubbles on tumoral endothelium. The presence of VEGFR2 was confirmed by immunofluorescence staining of tumor cryosections. CONCLUSIONS: VEGFR2-targeted ultrasound contrast agents such as BR55 will likely prove useful in human for the early detection of tumors as well as for the assessment of response to specific treatments.

Tuftsin binds neuropilin-1 through a sequence similar to that encoded by exon 8 of vascular endothelial growth factor.

Tuftsin, Thr-Lys-Pro-Arg (TKPR), is an immunostimulatory peptide with reported nervous system effects as well. We unexpectedly found that tuftsin and a higher affinity antagonist, TKPPR, bind selectively to neuropilin-1 and block vascular endothelial growth factor (VEGF) binding to that receptor. Dimeric and tetrameric forms of TKPPR had greatly increased affinity for neuropilin-1 based on competition binding experiments. On endothelial cells tetrameric TKPPR inhibited the VEGF(165)-induced autophosphorylation of vascular endothelial growth factor receptor-2 (VEGFR-2) even though it did not directly inhibit VEGF binding to VEGFR-2. Homology between exon 8 of VEGF and TKPPR suggests that the sequence coded for by exon 8 may stabilize VEGF binding to neuropilin-1 to facilitate signaling through VEGFR-2. Given the overlap between processes involving neuropilin-1 and tuftsin, we propose that at least some of the previously reported effects of tuftsin are mediated through neuropilin-1.