Zhuojun Wu

Image-guided, targeted and triggered drug delivery to tumors using polymer-based microbubbles

Microbubbles (MB) are routinely used contrast agents for functional and molecular ultrasound (US) imaging. In addition, they have been attracting more and more attention for drug delivery purposes, enabling e.g. US-mediated drug delivery across biological barriers and US-induced triggered drug release from the MB shell. The vast majority of efforts in this regard have thus far focused on phospholipid-based soft-shell MB, which are suboptimal for stably incorporating large amounts of drug molecules because of their relatively thin shell. Using poly(butyl cyanoacrylate) (PBCA)-based hard-shell MB, we show here that both hydrophilic (Rhodamine-B) and hydrophobic (Coumarin-6) model drugs can be efficiently and stably entrapped within the ~50 nm shell of PBCA MB. In addition, we demonstrate that model drug loading does not negatively affect the acoustic properties of the MB, and that functionalizing the surface of fluorophore-loaded MB with anti-VEGFR2 antibodies enables image-guided and targeted model drug delivery to tumor blood vessels. Finally, we show both in vitro and in vivo that disintegrating VEGFR2-targeted MB with high-mechanical index US pulses leads to high levels of model drug release. Consequently, these findings indicate that polymer-based MB are highly suitable systems for image-guided, targeted and triggered drug delivery to tumors and tumor blood vessels.

Activation of CXCR7 Limits Atherosclerosis and Improves Hyperlipidemia by Increasing Cholesterol Uptake in Adipose Tissue

Background— The aim of this study was to determine the role of the chemokine receptor CXCR7 in atherosclerosis and vascular remodeling. CXCR7 is the alternative receptor of CXCL12, which regulates stem cell–mediated vascular repair and limits atherosclerosis via its receptor, CXCR4. Methods and Results— Wire-induced injury of the carotid artery was performed in mice with a ubiquitous, conditional deletion of CXCR7 and in mice treated with the synthetic CXCR7 ligand CCX771. The effect of CCX771 treatment on atherosclerosis was studied in apolipoprotein E–deficient (Apoe−/−) mice fed a high-fat diet for 12 weeks. Lipoprotein fractions were quantified in the plasma of Apoe−/− mice by fast protein liquid chromatography. Uptake of DiI-labeled very low-density lipoprotein to adipose tissue was determined by 2-photon microscopy. We show that genetic deficiency of Cxcr7 increased neointima formation and lesional macrophage accumulation in hyperlipidemic mice after vascular injury. This was related to increased serum cholesterol levels and subsequent hyperlipidemia-induced monocytosis. Conversely, administration of the CXCR7 ligand CCX771 to Apoe−/− mice inhibited lesion formation and ameliorated hyperlipidemia after vascular injury and during atherosclerosis. Treatment with CCX771 reduced circulating very low-density lipoprotein levels but not low-density lipoprotein or high-density lipoprotein levels and increased uptake of very low-density lipoprotein into Cxcr7-expressing white adipose tissue. This effect of CCX771 was associated with an enhanced lipase activity and reduced expression of Angptl4 in adipose tissue. Conclusions— CXCR7 regulates blood cholesterol by promoting its uptake in adipose tissue. This unexpected cholesterol-lowering effect of CXCR7 is beneficial for atherosclerotic vascular diseases, presumably via amelioration of hyperlipidemia-induced monocytosis, and can be augmented with a synthetic CXCR7 ligand.

Ultrasound molecular imaging of E-selectin in tumor vessels using poly n-butyl cyanoacrylate microbubbles covalently coupled to a short targeting peptide.

ObjectivesThe purposes of this study were the development and preclinical evaluation of clinically translatable E-selectin–specific ultrasound contrast agents based on a peptide ligand with the recognition sequence IELLQAR. Materials and MethodsThe E-selectin–specific peptide was synthesized through solid phase peptide synthesis and covalently attached to poly n-butylcyanoacrylate–stabilized microbubbles with an air core. Quantification of the microbubble surface coverage with peptides was performed through flow cytometry. Targeted adhesion of peptide-coated microbubbles was investigated in vitro using parallel plate flow chamber assays on tumor necrosis factor-&agr;–stimulated human umbilical vein endothelial cells. In vivo imaging was performed in nude mice bearing human ovarian carcinoma xenografts (MLS), followed by ex vivo immunohistochemistry validation of E-selectin expression. ResultsSuccess of peptide synthesis was validated through preparative reverse phase high-pressure liquid chromatography and electronspray ionization-mass spectrometry. Results of the flow cytometry revealed approximately 4000 E-selectin–specific peptides/microbubble surface. Results of the in vitro experiments demonstrated the specificity of peptide-coated microbubbles to E-selectin (1.10 ± 0.48 vs 0.19 ± 0.09 bound microbubbles per cell, before and after competition respectively; P < 0.01). The in vivo imaging enabled specific assessment of E-selectin expression in MLS carcinoma xenografts (5.21 ± 3.41 vs 1.37 ± 0.67 contrast intensity before and after competition, respectively; P < 0.05). ConclusionsClinically translatable microbubbles that were covalently coupled to the short E-selectin–specific peptide (IELLQAR) enabled specific imaging of the E-selectin expression in tumor vessels in vivo.

Noninvasive Molecular Ultrasound Monitoring of Vessel Healing After Intravascular Surgical Procedures in a Preclinical Setup

Objective—Cardiovascular interventions induce damage to the vessel wall making antithrombotic therapy inevitable until complete endothelial recovery. Without a method to accurately determine the endothelial status, many patients undergo prolonged anticoagulation therapy, denying them any invasive medical procedures, such as surgical operations and dental interventions. Therefore, we aim to introduce molecular ultrasound imaging of the vascular cell adhesion molecule (VCAM)-1 using targeted poly-n-butylcyanoacrylate microbubbles (MBVCAM-1) as an easy accessible method to monitor accurately the reendothelialization of vessels. Approach and Results—ApoE−/− mice were fed with an atherogenic diet for 1 and 12 weeks and subsequently, endothelial denudation was performed in the carotid arteries using a guidewire. Molecular ultrasound imaging was performed at different time points after denudation (1, 3, 7, and 14 days). An increased MBVCAM-1 binding after 1 day, a peak after 3 days, and a decrease after 7 days was found. After 12 weeks of diet, MBVCAM-1 binding also peaked after 3 days but remained high until 7 days, indicating a delay in endothelial recovery. Two-photon laser scanning microscopy imaging of double fluorescence staining confirmed the exposure of VCAM-1 on the superficial layer after arterial injury only during the healing phase. After complete reendothelialization, VCAM-1 expression persisted in the subendothelial layer but was not reachable for the MBVCAM-1 anymore. Conclusion—Molecular ultrasound imaging with MBVCAM-1 is promising to assess vascular damage and to monitor endothelial recovery after arterial interventions. Thus, it may become an important diagnostic tool supporting the development of adequate therapeutic strategies to personalize anticoagulant and anti-inflammatory therapy after cardiovascular intervention.

Rhodamine-Loaded Intercellular Adhesion Molecule–1-targeted Microbubbles for Dual-Modality Imaging Under Controlled Shear Stresses

Background—The ability to image incipient atherosclerosis is based on the early events taking place at the endothelial level. We hypothesized that the expression of intercellular adhesion molecule-1 even in vessels with high flow rates can be imaged at the molecular level using 2 complementary imaging techniques: 2-photon laser scanning microscopy and contrast-enhanced ultrasound. Methods and Results—Using 2-photon laser scanning microscopy and contrast-enhanced ultrasound, intercellular adhesion molecule-1–targeted and rhodamine-loaded microbubbles were shown to be specifically bound to tumor necrosis factor-&agr;–stimulated human umbilical vein endothelial cells and murine carotid arteries (44 wild-type mice) at shear stresses ranging from 1.25 to 120 dyn/cm2. Intercellular adhesion molecule-1–targeted and rhodamine-loaded microbubbles bound 8× more efficient (P=0.016) to stimulated human umbilical vein endothelial cells than to unstimulated cells and 14× more than nontargeted microbubbles (P=0.016). In excised carotids, binding efficiency did not decrease significantly when increasing the flow rate from 0.25 to 0.6 mL/min. Higher flow rates (0.8 and 1 mL/min) showed significantly reduced microbubbles retention, by 38% (P=0.03) and 55% (P=0.03), respectively. Ex vivo results were translatable in vivo, confirming that intercellular adhesion molecule-1–targeted and rhodamine-loaded microbubbles are able to bind specifically to the inflamed carotid artery endothelia under physiological flow conditions and to be noninvasively detected using contrast-enhanced ultrasound. Conclusions—Our data provide groundwork for the implementation of molecular ultrasound imaging in vessels with high shear stress and flow rates, as well as for the future development of image-guided therapeutic interventions, and multiphoton microscopy as the appropriate method of validation.

Fluorescently labeled microbubbles for facilitating translational molecular ultrasound studies

Microbubbles (MB) are routinely used as contrast agents for functional and molecular ultrasound (US) imaging. For molecular US imaging, MB are functionalized with antibodies or peptides, in order to visualize receptor expression by angiogenic or inflamed endothelium. In general, initial in vitro binding studies with targeted MB are performed using phase contrast microscopy. Difficulties in the identification of MB in standard phase contrast microscopy, however, generally result in high variability, high observer dependency, and low reproducibility. To overcome these shortcomings, we here describe a simple post-loading strategy for labeling polymer-based MB with fluorophores, and we show that the use of rhodamine-loaded MB in combination with fluorescence microscopy substantially reduces the variability and the observer dependency of in vitro binding studies. In addition, we demonstrate that rhodamine-loaded MB can also be used for in vivo and ex vivo experimental setups, e.g., for analyzing MB binding to inflamed carotids using two-photon laser scanning microscopy, and for validating the binding of VEGFR2-targeted MB to tumor endothelium. These findings demonstrate that fluorescently labeled MB substantially facilitate translational molecular US studies, and they suggest that a similar synthetic strategy can be exploited for preparing drug-loaded MB, to enable image-guided, targeted, and triggered drug delivery to tumors and to sites of inflammation.

Fluorophore labeling of core-crosslinked polymeric micelles for multimodal in vivo and ex vivo optical imaging

AIM To enable multimodal in vivo and ex vivo optical imaging of the biodistribution and tumor accumulation of core-crosslinked polymeric micelles (CCPMs). MATERIALS & METHODS mPEG-b-p(HPMAm-Lac)-based polymeric micelles, core-crosslinked via cystamine and covalently labeled with two different fluorophores (Dy-676/488), were synthesized. The CCPMs were intravenously injected into CT26 tumor-bearing mice. RESULTS Upon intravenous injection, the CCPMs accumulated in CT26 tumors reasonably efficiently, with values reaching approximately 4%ID at 24 h. Ex vivo two-photon laser scanning microscopy confirmed efficient extravasation of the image-guided CCPMs out of tumor blood vessels and relatively deep penetration into the tumor interstitium. CONCLUSION CCPMs were labeled with multiple fluorophores, and the results obtained exemplify that combining several different in vivo and ex vivo optical imaging techniques is highly useful for analyzing the biodistribution and tumor accumulation of nanomedicines.

PBCA-based polymeric microbubbles for molecular imaging and drug delivery

ABSTRACT Microbubbles (MB) are routinely used as contrast agents for ultrasound (US) imaging. We describe different types of targeted and drug‐loaded poly(n‐butyl cyanoacrylate) (PBCA) MB, and demonstrate their suitability for multiple biomedical applications, including molecular US imaging and US‐mediated drug delivery. Molecular imaging of angiogenic tumor blood vessels and inflamed atherosclerotic endothelium is performed by modifying the surface of PBCA MB with peptides and antibodies recognizing E‐selectin and VCAM‐1. Stable and inertial cavitation of PBCA MB enables sonoporation and permeabilization of blood vessels in tumors and in the brain, which can be employed for direct and indirect drug delivery. Direct drug delivery is based on US‐induced release of (model) drug molecules from the MB shell. Indirect drug delivery refers to US‐ and MB‐mediated enhancement of extravasation and penetration of co‐administered drugs and drug delivery systems. These findings are in line with recently reported pioneering proof‐of‐principle studies showing the usefulness of (phospholipid) MB for molecular US imaging and sonoporation‐enhanced drug delivery in patients. They aim to exemplify the potential and the broad applicability of combining MB with US to improve disease diagnosis and therapy. Graphical abstract Figure. No caption available.

Multi-photon microscopy in cardiovascular research

Multiphoton laser scanning microscopy has proven profound value for ex vivo 3D histology and in vivo imaging of motionless tissue. The development of triggering systems and fast imaging methods, combined with advanced preparation procedures solved the challenging task of intravital imaging of the fast pulsating heart and major arteries in animals and further increased the popularity of intravital multiphoton imaging in cardiovascular research. This review article will highlight the potential of multiphoton microscopy for the visualization and characterization of dynamical and structural processes involved in cardiac and vascular diseases, both in an ex vivo and an intravital animal setting. Examples will be given how multiphoton microscopy can be applied to imaging of atherosclerotic plaque development and progression at subcellular level as well as to intravital imaging of inflammatory processes in the heart. In addition to highlighting the potential of multiphoton microscopy in preclinical cardiovascular research, we will discuss how this tool and its applications may be clinically translated to support disease diagnosis and therapy in patients.

Molecular Ultrasound Imaging of Junctional Adhesion Molecule A Depicts Acute Alterations in Blood Flow and Early Endothelial Dysregulation

Objective— The junctional adhesion molecule A (JAM-A) is physiologically located in interendothelial tight junctions and focally redistributes to the luminal surface of blood vessels under abnormal shear and flow conditions accompanying atherosclerotic lesion development. Therefore, JAM-A was evaluated as a target for molecularly targeted ultrasound imaging of transient endothelial dysfunction under acute blood flow variations. Approach and Results— Flow-dependent endothelial dysfunction was induced in apolipoprotein E–deficient mice (n=43) by carotid partial ligation. JAM-A expression was investigated by molecular ultrasound using antibody-targeted poly(n-butyl cyanoacrylate) microbubbles and validated with immunofluorescence. Flow disturbance and arterial remodeling were assessed using functional ultrasound. Partial ligation led to an immediate drop in perfusion at the ligated side and a direct compensatory increase at the contralateral side. This was accompanied by a strongly increased JAM-A expression and JAM-A–targeted microbubbles binding at the partially ligated side and by a moderate and temporary increase in the contralateral artery (≈14× [P<0.001] and ≈5× [P<0.001] higher than control, respectively), both peaking after 2 weeks. Subsequently, although JAM-A expression and JAM-A–targeted microbubbles binding persisted at a higher level at the partially ligated side, it completely normalized within 4 weeks at the contralateral side. Conclusions— Temporary blood flow variations induce endothelial rearrangement of JAM-A, which can be visualized using JAM-A–targeted microbubbles. Thus, JAM-A may be considered as a marker of acute endothelial activation and dysfunction. Its imaging may facilitate the early detection of cardiovascular risk areas, and it enables the therapeutic prevention of their progression toward an irreversible pathological state.