Aminolysis-mediated single-step surface functionalization of poly (butyl cyanoacrylate) microbubbles for ultrasound molecular imaging.

Molecular ultrasound imaging with actively targeted microbubbles (MB) proved promising in preclinical studies but its clinical translation is limited. To achieve this, it is essential that the actively targeted MB can be produced with high batch-to-batch reproducibility with a controllable and defined number of binding ligands on the surface. In this regard, poly (n-butyl cyanoacrylate) (PBCA)-based polymeric MB have been used for US molecular imaging, however, ligand coupling was mostly done via hydrolysis and carbodiimide chemistry, which is a multi-step procedure with poor reproducibility and low MB yield. Herein, we developed a single-step coupling procedure resulting in high MB yields with minimal batch-to-batch variation. Actively targeted PBCA-MB were generated using an aminolysis protocol, wherein amine-containing cRGD was added to the MB using lithium methoxide as a catalyst. We confirmed the successful conjugation of cRGD on the MB surface, while preserving their structure and acoustic signal. Compared to the conventional hydrolysis protocol, aminolysis resulted in higher MB yields and better reproducibility of coupling efficiency. Optical imaging revealed that under flow conditions, cRGD- and rhodamine-labelled MB, generated by aminolysis, specifically bind to tumor necrosis factor-alpha (TNF-α) activated endothelial cells in vitro. Furthermore, US molecular imaging demonstrated a markedly higher binding of the cRGD-MB than of control MB in TNF-α activated mouse aortas and 4T1 tumors in mice. Thus, using the aminolysis based conjugation approach, important refinements on the production of cRGD-MB could be achieved that will facilitate the production of clinical-scale formulations with excellent binding and ultrasound imaging performance.

Riboflavin-targeted polymers improve tolerance of paclitaxel while maintaining therapeutic efficacy.

Active targeting can enhance precision and efficacy of drug delivery systems (DDS) against cancers. Riboflavin (RF) is a promising ligand for active targeting due to its biocompatibility and high riboflavin-receptor expression in cancers. In this study, RF-targeted 4-arm polyethylene glycol (PEG) stars conjugated with Paclitaxel (PTX), named PEG PTX RF, were evaluated as a targeted DDS. In vitro, PEG PTX RF exhibited higher toxicity against tumor cells compared to the non-targeted counterpart (PEG PTX), while free PTX displayed the highest acute toxicity. In vivo, all treatments were similarly effective, but PEG PTX RF-treated tumors showed fewer proliferating cells, pointing to sustained therapy effects. Moreover, PTX-treated animals' body and liver weights were significantly reduced, whereas both remained stable in PEG PTX and PEG PTX RF-treated animals. Overall, our targeted and non-targeted DDS reduced PTX's adverse effects, with RF targeting promoted drug uptake in cancer cells for sustained therapeutic effect.

Welfare Assessment on Healthy and Tumor-Bearing Mice after Repeated Ultrasound Imaging.

INTRODUCTION: Ultrasound (US) imaging enables tissue visualization in high spatial resolution with short examination times. Thus, it is often applied in preclinical research. Diagnostic US, including contrast-enhanced US (CEUS), is considered to be well-tolerated by laboratory animals although no systematic study has been performed to confirm this claim. Therefore, the aim of this study was to screen for possible effects of US and CEUS examinations on welfare of healthy mice. Additionally, the potential influence of CEUS and molecular CEUS on well-being and therapy response to regorafenib was investigated in breast cancer-bearing mice. MATERIAL AND METHODS: Forty healthy Balb/c mice were randomly assigned for examination with US or CEUS (3×/week) for 4 weeks. Untreated healthy mice and mice receiving only isoflurane anesthesia served as controls (n = 10/group). Ninety-four 4T1 tumor-bearing Balb/c mice were allocated randomly to the following groups: no imaging, isoflurane anesthesia, CEUS, and molecular CEUS. They either received 10 mg/kg regorafenib or vehicle solution daily by oral gavage. Animals were examined three times within 2 weeks. CEUS measurements were performed using phospholipid microbubbles, and phospholipid microbubbles targeting the vascular endothelial growth factor receptor-2 were applied for molecular CEUS. Welfare evaluation was performed by daily observational score sheets, measuring the heart rate, Rotarod performance, and fecal corticosterone metabolites twice a week. On the last day, pathological changes in serum corticosterone concentrations, hemograms, and organ weights were obtained. Moreover, a potential influence of isoflurane anesthesia, CEUS, and molecular CEUS on regorafenib response in tumor-bearing mice was examined. Analysis of variance and Dunnett's post hoc test were performed as statistical analyses. RESULTS: Severity parameters were not altered after repeated US and CEUS examinations of healthy mice, but spleen sizes were significantly lower after isoflurane anesthesia. In tumor-bearing mice, no effect on animal welfare after repeated CEUS and molecular CEUS could be observed. However, leukocyte counts and spleen weights of tumor-bearing mice were significantly lower in animals examined with CEUS and molecular CEUS compared to the control groups. This effect was not visible in regorafenib-treated animals. CONCLUSIONS: Repeated US and (molecular) CEUS have no detectable impact on animal welfare in healthy and tumor-bearing mice. However, CEUS and molecular CEUS in combination with isoflurane anesthesia might attenuate immunological processes in tumor-bearing animals and may consequently affect responses to antitumor therapy.

Molecular Ultrasound Imaging Depicts the Modulation of Tumor Angiogenesis by Acetylsalicylic Acid.

Acetylsalicylic acid (ASA) is a well-established drug for heart attack and stroke prophylaxis. Furthermore, numerous studies have reported an anti-carcinogenic effect, but its exact mechanism is still unknown. Here, we applied VEGFR-2-targeted molecular ultrasound to explore a potential inhibitory effect of ASA on tumor angiogenesis in vivo. Daily ASA or placebo therapy was performed in a 4T1 tumor mouse model. During therapy, ultrasound scans were performed using nonspecific microbubbles (CEUS) to determine the relative intratumoral blood volume (rBV) and VEGFR-2-targeted microbubbles to assess angiogenesis. Finally, vessel density and VEGFR-2 expression were assessed histologically. CEUS indicated a decreasing rBV in both groups over time. VEGFR-2 expression increased in both groups up to Day 7. Towards Day 11, the binding of VEGFR-2-specific microbubbles further increased in controls, but significantly (p = 0.0015) decreased under ASA therapy (2.24 ± 0.46 au vs. 0.54 ± 0.55 au). Immunofluorescence showed a tendency towards lower vessel density under ASA and confirmed the result of molecular ultrasound. Molecular US demonstrated an inhibitory effect of ASA on VEGFR-2 expression accompanied by a tendency towards lower vessel density. Thus, this study suggests the inhibition of angiogenesis via VEGFR-2 downregulation as one of the anti-tumor effects of ASA.

Ultrasound Microbubbles for Diagnosis and Treatment of Cardiovascular Diseases.

Ultrasound (US) imaging of heart and major arteries and veins is among the most frequently used diagnostic techniques applied in humans. Conventional cardiovascular US sessions include anatomical B-mode and functional M-, pulsed-wave- and Doppler mode, which have their limitations in both precise cardiac chambers' delineation and small vessel imaging. The introduction of contrast-enhanced US, employing microbubble suspensions as contrast agent, has enabled a better delineation of heart chambers, the visualization of myocardial microvasculature, and the atherosclerotic plaque neovascularization. Moreover, specific disease-related molecular tracers have been developed by modifying the microbubbles with targeting ligands directed to biological markers exposed to the luminal side of the blood vessels. Microbubble functionalization has enabled in vivo molecular US imaging of various stages of atherosclerosis, from plaque initiation to plaque vulnerability, and neointima formation following revascularization procedures. Furthermore, oscillating microbubbles have been used to mechanically dissolve thrombus material and may act as carriers of drugs and nucleic acids that are released locally by US pulses. This review article summarizes recent advances in functional and molecular US images and discusses therapeutic applications of microbubbles. The addressed topics include an overview on microbubble formats, microbubble detection methods, molecular targets of cardiovascular diseases, and the use of microbubbles for thrombolysis and drug delivery.

Molecular Ultrasound Imaging.

Contrast-enhanced ultrasound (CEUS) imaging is a valuable tool for preclinical and clinical diagnostics. The most frequently used ultrasound contrast agents are microbubbles. Besides them, novel nano-sized materials are under investigation, which are briefly discussed in this chapter. For molecular CEUS, the ultrasound contrast agents are modified to actively target disease-associated molecular markers with a site-specific ligand. The most common markers for tumor imaging are related to neoangiogenesis, like the vascular endothelial growth factor receptor-2 (VEGFR2) and αvß3 integrin. In this chapter, applications of molecular ultrasound to longitudinally monitor receptor expression during tumor growth, to detect neovascularization, and to evaluate therapy responses are described. Furthermore, we report on first clinical trials of molecular CEUS with VEGFR2-targeted phospholipid microbubbles showing promising results regarding patient safety and its ability to detect tumors of prostate, breast, and ovary. The chapter closes with an outlook on ultrasound theranostics, where (targeted) ultrasound contrast agents are used to increase the permeability of tumor tissues and to support drug delivery.

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.

A water-soluble PEGylated RGD-functionalized bisbithiophenyl diketopyrrolopyrrole as a photoacoustic sonophore.

Photoacoustic imaging presents an innocuous imaging modality with good penetration depth and resolution. To use this modality for detection and imaging of pathological sites, new imaging probes need to be developed to enhance the contrast over endogenous sonophores. These contrast agents should specifically bind to the site of interest, be non-toxic and be cleared renally if applied intravenously. Small organic dyes with absorption in the near infrared spectrum often exhibit good photoacoustic response. However, such dyes are often not water soluble or they are cytotoxic. Here, we present a novel PEGylated sonophore based on diketopyrrolopyrrole (DPP), which overcomes these limitations and can be functionalized with desired biological recognition motifs using thiol-yne click chemistry. Proof of concept is demonstrated by functionalizing the DPP-based probe with an RGD peptide, resulting in specific binding to endothelial (HUVEC) cells and an efficient photoacoustic response.

Balancing Passive and Active Targeting to Different Tumor Compartments Using Riboflavin-Functionalized Polymeric Nanocarriers.

Riboflavin transporters (RFTs) and the riboflavin carrier protein (RCP) are highly upregulated in many tumor cells, tumor stem cells, and tumor neovasculature, which makes them attractive targets for nanomedicines. Addressing cells in different tumor compartments requires drug carriers, which are not only able to accumulate via the EPR effect but also to extravasate, target specific cell populations, and get internalized by cells. Reasoning that antibodies are among the most efficient targeting systems developed by nature, we consider their size (∼10-15 nm) to be ideal for balancing passive and active tumor targeting. Therefore, small, short-circulating (10 kDa, ∼7 nm, t1/2 ∼ 1 h) and larger, longer-circulating (40 kDa, ∼13 nm, t1/2 ∼ 13 h) riboflavin-targeted branched PEG polymers were synthesized, and their biodistribution and target site accumulation were evaluated in mice bearing angiogenic squamous cell carcinoma (A431) and desmoplastic prostate cancer (PC3) xenografts. The tumor accumulation of the 10 kDa PEG was characterized by rapid intercompartmental exchange and significantly improved upon active targeting with riboflavin (RF). The 40 kDa PEG accumulated in tumors four times more efficiently than the small polymer, but its accumulation did not profit from active RF-targeting. However, RF-targeting enhanced the cellular internalization in both tumor models and for both polymer sizes. Interestingly, the nanocarriers' cell-uptake in tumors was not directly correlated with the extent of accumulation. For example, in both tumor models the small RF-PEG accumulated much less strongly than the large passively targeted PEG but showed significantly higher intracellular amounts 24 h after iv administration. Additionally, the size of the polymer determined its preferential uptake by different tumor cell compartments: the 10 kDa RF-PEGs most efficiently targeted cancer cells, whereas the highest uptake of the 40 kDa RF-PEGs was observed in tumor-associated macrophages. These findings imply that drug carriers with sizes in the range of therapeutic antibodies show balanced properties with respect to passive accumulation, tissue penetration, and active targeting. Besides highlighting the potential of RF-mediated (cancer) cell targeting, we show that strong tumor accumulation does not automatically mean high cellular uptake and that the nanocarriers' size plays a critical role in cell- and compartment-specific drug targeting.

Squamous Cell Carcinoma Xenografts: Use of VEGFR2-targeted Microbubbles for Combined Functional and Molecular US to Monitor Antiangiogenic Therapy Effects.

PURPOSE: To assess the ability of vascular endothelial growth factor receptor type 2 (VEGFR2)-targeted and nontargeted ultrasonography (US) to depict antiangiogenic therapy effects and to investigate whether first-pass kinetics obtained with VEGFR2-targeted microbubbles provide independent data about tumor vascularization. MATERIALS AND METHODS: Governmental approval was obtained for animal experiments. Vascularization in response to anti-vascular endothelial growth factor receptor or vehicle-control treatment (10 per group) in HaCaT-ras A-5RT3 xenografts was longitudinally assessed in mice by means of first-pass kinetics of nontargeted microbubbles (BR1, BR38; Bracco, Geneva, Switzerland) and VEGFR2-targeted microbubbles (BR55, Bracco) before and 4, 7, and 14 days after therapy. VEGFR2 expression was determined 8 minutes after BR55 injection with destruction-replenishment analysis. US data were validated with immunohistochemistry. Significant differences were evaluated with the Mann-Whitney test. RESULTS: First-pass analysis with BR1, BR38, and BR55 showed similar tendencies toward decreasing vascularization, with a stronger decrease in tumors treated with anti-VEGF antibody. The median signal intensity (in arbitrary units [au]) of anti-VEGF antibody-treated versus control tumors at day 14 was as follows: BR1, 5.2 au (interquartile range [IQR], 3.2 au) vs 11.3 au (IQR, 10.0 au), respectively; BR38, 6.2 au (IQR, 3.5) vs 10.0 au (IQR, 7.8); and BR55, 9.5 au (IQR, 6.0 au) vs 13.8 au (IQR, 9.8) (P = .0230). VEGFR2 assessment with BR55 demonstrated significant differences between both groups throughout the therapy period (median signal intensity of anti-VEGF antibody-treated vs control tumors: 0.04 au [IQR, 0.1 au] vs 0.14 au [IQR, 0.08 au], respectively, at day 4, P = .0058; 0.04 au [IQR, 0.06 au] vs 0.13 au [IQR, 0.09 au] at day 7, P = .0058; and 0.06 au [IQR, 0.11 au] vs 0.16 au [IQR, 0.15 au] at day 14, P = .0247). Immunohistochemistry confirmed the lower microvessel density and VEGFR2-positive area fraction in tumors treated with anti-VEGF antibody. CONCLUSION: Antiangiogenic therapy effects were detected earlier and more distinctly with VEGFR2-targeted US than with functional US. First-pass analyses with BR55, BR38, and BR1 revealed similar results, with a decrease in vascularization during therapy. Functional data showed that BR55 is not strongly affected by early binding of the microbubbles to VEGFR2. Thus, functional and molecular imaging of angiogenesis can be performed with BR55 within one examination.

In-vivo detection of the erythropoietin receptor in tumours using positron emission tomography.

OBJECTIVE: Recombinant human erythropoietin (rhuEpo) is used clinically to treat anaemia. However, rhuEpo-treated cancer patients show decreased survival rates and erythropoietin receptor (EpoR) expression has been found in patient tumour tissue. Thus, rhuEpo application might promote EpoR(+) tumour progression. We therefore developed the positron emission tomography (PET)-probe (68)Ga-DOTA-rhuEpo and evaluated its performance in EpoR(+) A549 non-small-cell lung cancer (NSCLC) xenografts. METHODS: (68)Ga-DOTA-rhuEpo was generated by coupling DOTA-hydrazide to carbohydrate side-chains of rhuEpo. Biodistribution was determined in tumour-bearing mice 0.5, 3, 6, and 9 h after probe injection. Competition experiments were performed by co-injecting (68)Ga-DOTA-rhuEpo and rhuEpo in five-fold excess. Probe specificity was further evaluated histologically using Epo-Cy5.5 stainings. RESULTS: The blood half-life of (68)Ga-DOTA-rhuEpo was 2.6 h and the unbound fraction was cleared by the liver and kidney. After 6 h, the highest tumour to muscle ratio was reached. The highest (68)Ga-DOTA-rhuEpo accumulation was found in liver (10.06 ± 6.26%ID/ml), followed by bone marrow (1.87 ± 0.53%ID/ml), kidney (1.58 ± 0.39%ID/ml), and tumour (0.99 ± 0.16%ID/ml). EpoR presence in these organs was histologically confirmed. Competition experiments showed significantly (p < 0.05) lower PET-signals in tumour and bone marrow at 3 and 6 h. CONCLUSION: (68)Ga-DOTA-rhuEpo shows favourable pharmacokinetic properties and detects EpoR specifically. Therefore, it might become a valuable radiotracer to monitor EpoR status in tumours and support decision-making in anaemia therapy. KEY POINTS: • PET-probe (68) Ga-DOTA-rhuEpo was administered to assess the EpoR status in vivo • (68) Ga-DOTA-rhuEpo binds specifically to EpoR positive organs in vivo • Tumour EpoR status determination might enable decision-making in anaemia therapy with rhuEpo.

A low molecular weight zinc2+-dipicolylamine-based probe detects apoptosis during tumour treatment better than an annexin V-based probe.

OBJECTIVES: Molecular imaging of apoptosis is frequently discussed for monitoring cancer therapies. Here, we compare the low molecular weight phosphatidylserine-targeting ligand zinc2+-dipicolylamine (Zn2+-DPA) with the established but reasonably larger protein annexin V. METHODS: Molecular apoptosis imaging with the fluorescently labelled probes annexin V (750 nm, 36 kDa) and Zn2+-DPA (794 nm, 1.84 kDa) was performed in tumour-bearing mice (A431). Three animal groups were investigated: untreated controls and treated tumours after 1 or 4 days of anti-angiogenic therapy (SU11248). Additionally, µPET with 18 F-FDG was performed. Imaging data were displayed as tumour-to-muscle ratio (TMR) and validated by quantitative immunohistochemistry. RESULTS: Compared with untreated control tumours, TUNEL staining indicated significant apoptosis after 1 day (P < 0.05) and 4 days (P < 0.01) of treatment. Concordantly, Zn2+-DPA uptake increased significantly after 1 day (P < 0.05) and 4 days (P < 0.01). Surprisingly, annexin V failed to detect significant differences between control and treated animals. Contrary to the increasing uptake of Zn2+-DPA, 18 F-FDG tumour uptake decreased significantly at days 1 (P < 0.05) and 4 (P < 0.01). CONCLUSIONS: Increase in apoptosis during anti-angiogenic therapy was detected significantly better with the low molecular weight probe Zn2+-DPA than with the annexin V-based probe. Additionally, significant treatment effects were detectable as early using Zn2+-DPA as with measurements of the glucose metabolism using 18 F-FDG. KEY POINTS: • The detection of apoptosis by non-invasive imaging is important in oncology. • A new low molecular weight probe Zn2+-DPA shows promise in depicting anti-angiogenic effects. • The small Zn2+-DPA ligand appears well suited for monitoring therapy. • Treatment effects are detectable just as early with Zn2+-DPA as with 18F-FDG.

Ultrasound-induced immune responses in tumors: A systematic review and meta-analysis.

Ultrasound is widely used in the diagnosis and therapy of cancer. Tumors can be treated by thermal or mechanical tissue ablation. Furthermore, tumors can be manipulated by hyperthermia, sonodynamic therapy and sonoporation, e.g., by increasing tumor perfusion or the permeability of biological barriers to enhance drug delivery. These treatments induce various immune responses in tumors. However, conflicting data and high heterogeneity between experimental settings make it difficult to generalize the effects of ultrasound on tumor immunity. Therefore, we performed a systematic review to answer the question: "Does ultrasound alter the immune reaction of peripheral solid tumors in humans and animals compared to control conditions without ultrasound?" A systematic literature search was performed in PubMed, EMBASE, and Web of Science and 24,401 potentially relevant publications were identified. Of these, 96 publications were eligible for inclusion in the systematic review. Experiments were performed in humans, rats, and mice and focused on different tumor types, primarily breast and melanoma. We collected data on thermal and non-thermal ultrasound settings, the use of sono-sensitizers or sono-enhancers, and anti-tumor therapies. Six meta-analyses were performed to quantify the effect of ultrasound on tumor infiltration by T cells (cytotoxic, helper, and regulatory T cells) and on blood cytokines (interleukin-6, interferon-γ, tumor necrosis factor-α). We provide robust scientific evidence that ultrasound alters T cell infiltration into tumors and increases blood cytokine concentrations. Furthermore, we identified significant differences in immune cell infiltration based on tumor type, ultrasound settings, and mouse age. Stronger effects were observed using hyperthermia in combination with sono-sensitizers and in young mice. The latter may impair the translational impact of study results as most cancer patients are older. Thus, our results may help refining ultrasound parameters to enhance anti-tumor immune responses for therapeutic use and to minimize immune effects in diagnostic applications.

mRNA Sonotransfection of Tumors with Polymeric Microbubbles: Co-Formulation versus Co-Administration.

Despite its high potential, non-viral gene therapy of cancer remains challenging due to inefficient nucleic acid delivery. Ultrasound (US) with microbubbles (MB) can open biological barriers and thus improve DNA and mRNA passage. Polymeric MB are an interesting alternative to clinically used lipid-coated MB because of their high stability, narrow size distribution, and easy functionalization. However, besides choosing the ideal MB, it remains unclear whether nanocarrier-encapsulated mRNA should be administered separately (co-administration) or conjugated to MB (co-formulation). Therefore, the impact of poly(n-butyl cyanoacrylate) MB co-administration with mRNA-DOTAP/DOPE lipoplexes or their co-formulation on the transfection of cancer cells in vitro and in vivo is analyzed. Sonotransfection improved mRNA delivery into 4T1 breast cancer cells in vitro with co-administration being more efficient than co-formulation. In vivo, the co-administration sonotransfection approach also resulted in higher transfection efficiency and reached deeper into the tumor tissue. On the contrary, co-formulation mainly promoted transfection of endothelial and perivascular cells. Furthermore, the co-formulation approach is much more dependent on the US trigger, resulting in significantly lower off-site transfection. Thus, the findings indicate that the choice of co-administration or co-formulation in sonotransfection should depend on the targeted cell population, tolerable off-site transfection, and the therapeutic purpose.

RGD-coated polymeric microbubbles promote ultrasound-mediated drug delivery in an inflamed endothelium-pericyte co-culture model of the blood-brain barrier.

Drug delivery to central nervous pathologies is compromised by the blood-brain barrier (BBB). A clinically explored strategy to promote drug delivery across the BBB is sonopermeation, which relies on the combined use of ultrasound (US) and microbubbles (MB) to induce temporally and spatially controlled opening of the BBB. We developed an advanced in vitro BBB model to study the impact of sonopermeation on the delivery of the prototypic polymeric drug carrier pHPMA as a larger molecule and the small molecule antiviral drug ribavirin. This was done under standard and under inflammatory conditions, employing both untargeted and RGD peptide-coated MB. The BBB model is based on human cerebral capillary endothelial cells and human placental pericytes, which are co-cultivated in transwell inserts and which present with proper transendothelial electrical resistance (TEER). Sonopermeation induced a significant decrease in TEER values and facilitated the trans-BBB delivery of fluorescently labeled pHPMA (Atto488-pHPMA). To study drug delivery under inflamed endothelial conditions, which are typical for e.g. tumors, neurodegenerative diseases and CNS infections, tumor necrosis factor (TNF) was employed to induce inflammation in the BBB model. RGD-coated MB bound to and permeabilized the inflamed endothelium-pericyte co-culture model, and potently improved Atto488-pHPMA and ribavirin delivery. Taken together, our work combines in vitro BBB bioengineering with MB-mediated drug delivery enhancement, thereby providing a framework for future studies on optimization of US-mediated drug delivery to the brain.

Polymeric Microbubble Shell Engineering: Microporosity as a Key Factor to Enhance Ultrasound Imaging and Drug Delivery Performance.

Microbubbles (MB) are widely used as contrast agents for ultrasound (US) imaging and US-enhanced drug delivery. Polymeric MB are highly suitable for these applications because of their acoustic responsiveness, high drug loading capability, and ease of surface functionalization. While many studies have focused on using polymeric MB for diagnostic and therapeutic purposes, relatively little attention has thus far been paid to improving their inherent imaging and drug delivery features. This study here shows that manipulating the polymer chemistry of poly(butyl cyanoacrylate) (PBCA) MB via temporarily mixing the monomer with the monomer-mimetic butyl cyanoacetate (BCC) during the polymerization process improves the drug loading capacity of PBCA MB by more than twofold, and the in vitro and in vivo acoustic responses of PBCA MB by more than tenfold. Computer simulations and physisorption experiments show that BCC manipulates the growth of PBCA polymer chains and creates nanocavities in the MB shell, endowing PBCA MB with greater drug entrapment capability and stronger acoustic properties. Notably, because BCC can be readily and completely removed during MB purification, the resulting formulation does not include any residual reagent beyond the ones already present in current PBCA-based MB products, facilitating the potential translation of next-generation PBCA MB.

In vitro and in vivo evaluation of biohybrid tissue-engineered vascular grafts with transformative 1H/19F MRI traceable scaffolds.

Biohybrid tissue-engineered vascular grafts (TEVGs) promise long-term durability due to their ability to adapt to hosts' needs. However, the latter calls for sensitive non-invasive imaging approaches to longitudinally monitor their functionality, integrity, and positioning. Here, we present an imaging approach comprising the labeling of non-degradable and degradable TEVGs' components for their in vitro and in vivo monitoring by hybrid 1H/19F MRI. TEVGs (inner diameter 1.5 mm) consisted of biodegradable poly(lactic-co-glycolic acid) (PLGA) fibers passively incorporating superparamagnetic iron oxide nanoparticles (SPIONs), non-degradable polyvinylidene fluoride scaffolds labeled with highly fluorinated thermoplastic polyurethane (19F-TPU) fibers, a smooth muscle cells containing fibrin blend, and endothelial cells. 1H/19F MRI of TEVGs in bioreactors, and after subcutaneous and infrarenal implantation in rats, revealed that PLGA degradation could be faithfully monitored by the decreasing SPIONs signal. The 19F signal of 19F-TPU remained constant over weeks. PLGA degradation was compensated by cells' collagen and α-smooth-muscle-actin deposition. Interestingly, only TEVGs implanted on the abdominal aorta contained elastin. XTT and histology proved that our imaging markers did not influence extracellular matrix deposition and host immune reaction. This concept of non-invasive longitudinal assessment of cardiovascular implants using 1H/19F MRI might be applicable to various biohybrid tissue-engineered implants, facilitating their clinical translation.

Liver dysplasia: US molecular imaging with targeted contrast agent enables early assessment.

PURPOSE: To investigate the ability of vascular endothelial growth factor receptor type 2 (VEGFR2)-targeted ultrasonographic (US) microbubbles for the assessment of liver dysplasia in transgenic mice. MATERIALS AND METHODS: Animal experiments were approved by the governmental review committee. Nuclear factor-κB essential modulator knock-out mice with liver dysplasia and wild-type mice underwent liver imaging by using a clinical US system. Two types of contrast agents were investigated: nontargeted, commercially available, second-generation microbubbles (SonoVue) and clinically translatable PEGylated VEGFR2-targeted microbubbles (BR55). Microbubble kinetics was investigated over the course of 4 minutes. Targeted contrast material-enhanced US signal was quantified 5 minutes after injection. Competitive in vivo binding experiments with BR55 were performed in knock-out mice. Immunohistochemical and hematoxylin-eosin staining of liver sections was performed to validate the in vivo US results. Groups were compared by using the Mann-Whitney test. RESULTS: Peak enhancement after injection of SonoVue and BR55 did not differ in healthy and dysplastic livers (SonoVue, P = .46; BR55, P = .43). Accordingly, immunohistochemical findings revealed comparable vessel densities in both groups. The specificity of BR55 to VEGFR2 was proved by in vivo competition (P = .0262). While the SonoVue signal decreased similarly in healthy and dysplastic livers during the 4 minutes, there was an accumulation of BR55 in dysplastic livers compared with healthy ones. Furthermore, targeted contrast-enhanced US signal indicated a significantly higher site-specific binding of BR55 in dysplastic than healthy livers (P = .005). Quantitative immunohistologic findings confirmed significantly higher VEGFR2 levels in dysplastic livers (P = .02). CONCLUSION: BR55 enables the distinction of early stages of liver dysplasia from normal liver.

The high angiogenic activity in very early breast cancer enables reliable imaging with VEGFR2-targeted microbubbles (BR55).

OBJECTIVES: Tumour xenografts of well-discernible sizes can be examined well by molecular ultrasound. Here, we investigated whether very early breast carcinomas express sufficient levels of VEGFR2 for reliable molecular ultrasound imaging with targeted microbubbles. METHODS: MCF-7 breast cancer xenografts were orthotopically implanted in nude mice (n = 26). Tumours measuring from 4 mm(3) (2 mm diameter) up to 65 mm(3) (5 mm diameter) were examined with automated 3D molecular ultrasound using clinically translatable VEGFR2-targeted microbubbles (BR55). Additionally, the relative tumour blood volume was assessed with non-targeted microbubbles (BR38). In vivo ultrasound data were validated by quantitative immunohistochemistry. RESULTS: Very small lesions 2 mm in diameter showed the highest binding of VEGFR2-specific microbubbles. In larger tumours significantly less BR55 accumulated (p = 0.023). Nonetheless, binding of VEGFR2-targeted microbubbles was still high enough for imaging. The relative blood volume was comparable at all tumour sizes. Both findings were confirmed by immunohistochemistry. Additionally, a significantly enhanced number of large and mature vessels were detected with increasing tumour size (p < 0.01), explaining the decrease in VEGFR2 expression during tumour growth. CONCLUSIONS: 3D molecular ultrasound using BR55 is very well suited to depicting the angiogenic activity in very small breast lesions, suggesting its potential for detecting and characterising these lesions.

Repeated Contrast-Enhanced Micro-CT Examinations Decrease Animal Welfare and Influence Tumor Physiology.

OBJECTIVES: Computed tomography (CT) imaging is considered relatively safe and is often used in preclinical research to study physiological processes. However, the sum of low-dose radiation, anesthesia, and animal handling might impact animal welfare and physiological parameters. This is particularly relevant for longitudinal studies with repeated CT examinations. Therefore, we investigated the influence of repeated native and contrast-enhanced (CE) CT on animal welfare and tumor physiology in regorafenib-treated and nontreated tumor-bearing mice. MATERIAL AND METHODS: Mice bearing 4T1 breast cancer were divided into 5 groups: (1) no imaging, (2) isoflurane anesthesia only, (3) 4 mGy CT, (4) 50 mGy CT, and (5) CE-CT (iomeprol). In addition, half of each group was treated with the multikinase inhibitor regorafenib. Mice were imaged 3 times within 1 week under isoflurane anesthesia. Behavioral alterations were investigated by score sheet evaluation, rotarod test, heart rate measurements, and fecal corticosterone metabolite analysis. Tumor growth was measured daily with a caliper. Tumors were excised at the end of the experiment and histologically examined for blood vessel density, perfusion, and cell proliferation. RESULTS: According to the score sheet, animals showed a higher burden after anesthesia administration and in addition with CT imaging ( P < 0.001). Motor coordination was not affected by native CT, but significantly decreased after CE-CT in combination with the tumor therapy ( P < 0.001). Whereas tumor growth and blood vessel density were not influenced by anesthesia or imaging, CT-scanned animals had a higher tumor perfusion ( P < 0.001) and a lower tumor cell proliferation ( P < 0.001) for both radiation doses. The most significant difference was observed between the control and CE-CT groups. CONCLUSION: Repeated (CE-) CT imaging of anesthetized animals can lead to an impairment of animal motor coordination and, thus, welfare. Furthermore, these standard CT protocols seem to be capable of inducing alterations in tumor physiology when applied repetitively. These potential effects of native and CE-CT should be carefully considered in preclinical oncological research.