Molecular ultrasound imaging was proposed more than two decades ago. The ultrasound contrast agents used for selective targeting of vascular biomarkers of disease are formulated as gas microbubbles surrounded by a thin shell.Microbubbles are decorated by the ligands that specifically bind to the biomarkers of disease overexpressed on the surface of vascular endothelium. The microbubble gas core is most often a poorly soluble perfluorinated gas (preferably C4F10 as the least soluble), to assure circulation lifetime for at least several minutes. The gas from circulating bubbles is exhaled via the lungs; bubbles collapse and lose echogenicity. As the circulating bubbles clear from the bloodstream, adherent targeted bubbles are still retained in the tumor microvasculature and provide strong acoustic backscatter and ultrasound detection capability. A critical feature that enables ultrasound use in molecular imaging, specifically in the oncology setting, is the detection sensitivity, already enabled in medical imaging equipment. Individual microbubbles, with sub-picogram mass, can be visualized in real time, many cm deep in the body. This chapter discusses microbubble preparation and attachment of targeting ligands to the microbubble shell. The logic of the performance of ultrasound molecular imaging is discussed, followed by an assessment of a number of imaging tools.

Normal prostate epithelial cells accumulate high intracellular zinc levels that maintain optimum mitochondrial metabolism and proliferation. Prostate cancer cells lose this zinc-accumulating capacity, enabling metabolic reprogramming that supports tumor progression. Restoring intracellular zinc selectively in prostate tumors represents a promising therapeutic strategy; however, systemic zinc administration is limited by the inability of prostate cancer cells to take up free zinc resulting from ZIP1 transporter downregulation. To overcome this challenge, we developed a formulation of prostate-specific membrane antigen (PSMA)-targeted, zinc-loaded liposomes (Zn-TL) to enable tumor-selective intracellular zinc delivery. Zn-TL was prepared with uniform nanoscale size, low polydispersity, and negative surface charge. The formulation showed minimal zinc leakage during storage and sustained retention in vitro. In prostate cancer cells, Zn-TL demonstrated receptor-mediated uptake, resulting in increased cytotoxicity and apoptosis. In vivo, we performed proof-of-principle studies showing prolonged circulation and tumor accumulation of Zn-TL in mice bearing PSMA-positive tumors. While tumor growth was delayed during early and intermediate stages of tumor development, this effect diminished at later stages. The stage-dependent efficacy suggests that Zn-TL may be most effective when used earlier in disease progression. These results also suggest that Zn-TL represents a promising platform for metabolic intervention and may benefit from combination strategies to enhance efficacy in advanced disease.

Glioblastoma is a fatal primary malignant brain tumor, with an average survival of 15 months despite surgical resection, chemotherapy, and radiation therapy. Due to the concurrent deregulation of numerous genes in glioblastoma, molecular monotherapies have not improved clinical outcomes. Evidence suggests that targeting multiple deregulated molecules is essential for better therapies; however, this is limited by the lack of suitable drugs and increased toxicity of combination therapies. To address this, we hypothesized that miRNAs, small gene-regulatory RNAs that suppress mRNA, could simultaneously inhibit multiple deregulated genes in glioblastoma and be used for more effective therapies. We identified regulatory miRNAs — those that target several deregulated genes in glioblastoma — using a combination of PAR-CLIP screening, TCGA data analyses, and an algorithm to rank target importance and miRNA therapeutic potential. We selected 2 tumor-suppressive miRNAs, miR-340 and miR-382, and 1 oncogenic miRNA, miR-17, and showed that they targeted critical glioblastoma pathways and altered cell growth, survival, invasion, and in vivo tumor growth. We developed and successfully applied a miRNA therapeutic delivery approach using brain-penetrating nanoparticles combined with MRI-guided focused ultrasound and microbubbles, to inhibit established tumor growth and extend animal survival. This strategy offers a promising approach for translating miRNA-based therapies into clinical trials for glioblastoma and other cancers.

Ultrasound localization microscopy (ULM) enables super-resolution ultrasound (SRUS) imaging of microvasculature, while ultrasound molecular imaging (USMI) characterizes molecular signatures using microbubbles (MBs) targeted to specific biomarkers. Although the co-localization of SRUS and USMI has been demonstrated previously, USMI resolution is limited by ultrasound diffraction-based effects and does not match the super-resolved microvasculature. This study introduces the Incremental Burst Sequence (IBS) method to induce the population of polydisperse targeted MBs to burst progressively, achieving MBs spatial separation and enabling high-resolution USMI (HR-USMI) localization. IBS method employs interleaved imaging and bursting pulses, with transmit voltages of bursting pulses incrementally increased to produce a gradual rise in the acoustic pressure. IBS is first validated optically in vitro using a cellulose tubing phantom, and MB remaining count during IBS is measured. Thereafter, in vivo validation is performed in a murine tumor model, and the intra-tumoral targeted MB signal intensity is measured during IBS. Furthermore, high frame-rate data for SRUS and IBS data for HR-USMI are acquired from a single bolus injection of MBs to generate composite images with high-resolution molecular signatures superimposed on the tumor microvasculature. Both in vitro and in vivo results validate the technical feasibility of the proposed IBS method. In addition, we demonstrate that higher bursting pulse repetitions lead to a faster disruption of the MB population during IBS. Finally, HR-USMI signals localized within a 50 μm × 50 μm grid are aligned with microvessels resolved better than 100 μm, presenting a combination of molecular signatures and anatomical structures at fine resolution.

Objective: Mouse models of hindlimb ischemia (HLI) are used to study peripheral arterial disease and evaluate novel therapies. Contrast-enhanced ultrasound (CEUS) is a noninvasive perfusion measurement technique that is increasingly being employed in these models. The objective of this study was to evaluate two models of severe HLI by CEUS to characterize perfusion recovery and muscle perfusion patterns. Methods: Mice undergoing double femoral artery ligation were measured by CEUS and laser Doppler perfusion imaging (LDPI) at baseline and 1-150 d postsurgery. A second group undergoing femoral artery ligation and excision was measured 1-28 d postsurgery. Results: By LDPI, both surgeries showed robust perfusion recovery by 14 d postsurgery. However, by CEUS only a similar to 40% perfusion recovery plateau was reached in either group. These results are consistent with our previous work, employing a less severe single femoral artery ligation, that showed perfusion in the ischemic limb does not return to normal by 150 d postsurgery. Cluster analysis of muscle perfusion patterns indicated 3-5 different patterns at day 1 postsurgery. The double ligation model yielded significantly less variable perfusion patterns, suggesting that it can provide more reproducible results. Conclusion: Contrary to LDPI, perfusion as measured by CEUS never fully recovers after hindlimb surgery, even when followed 28-150 d postsurgery. Individual mice can manifest different patterns of muscle perfusion to the same surgery, but these patterns are conserved within and between different surgical techniques. These results may have significant implications for the evaluation of novel therapeutics to treat PAD in mice.

BACKGROUND:Glioblastoma (GBM) is a rapidly growing, aggressive brain tumor with very poor prognosis without currently effective therapies. The immunosuppressive nature of the tumor microenvironment (TME) in GBM hinders the development of effective tumor-eradicating immunotherapies. This hostile TME can be modulated by administering immune-activating cytokines in combination with agents inducing tumor cell death. To achieve these objectives, we sought to harness the cancer-selective cell death-inducing properties of an enhanced "Superkine" version of melanoma differentiation associated gene-7/interleukin-24, IL-24S, and the immune-activating properties of IL-15 to modulate the TME of GBM to maximize therapeutic outcomes. METHODS:A fusion "Superkine" (FSK) comprised of IL-24S linked to IL-15 was generated, and antitumor effects were evaluated when transduced by a type 5 adenovirus (Ad.5) in a GBM immunocompetent mouse tumor model. To target the delivery of Ad.5 FSK systemically, we employed an innovative approach of focused ultrasound (FUS) paired with microbubbles (MBs), FUS-DMB (FUS plus double MB), to safely transport the FSK engineered Ad.5 construct into mouse brain to overcome limitations of systemic viral delivery and selectivity of the blood-brain barrier. RESULTS:The FSK stimulated higher tumor regression and enhanced survival in vivo than the individual "Superkine" or cytokine in GBM cancer models. Apoptosis of GBM cells was induced, as well as increased tumor infiltration of T cells, dendritic cells, macrophages and natural killer (NK) cells. The antitumor-inducing activity of FSK is a consequence of induction of cancer-specific growth suppression and induction of apoptosis (IL-24S) as well as diverse effects on immune cells (IL-15 and IL-24S). Antibody neutralization indicates that a primary immune mediator of anticancer activity of FSK is through recruitment and activation of NK cells. Global cytokine analyses indicated no changes in inflammatory cytokines during therapy, suggesting that this strategy will be safe. CONCLUSION:In summary, treatment with an FSK, consisting of a fusion of IL-24S to IL-15, promotes GBM cell killing and remodeling of the TME by recruiting and activating immune cells supporting the feasibility of developing safe and effective cancer immunotherapeutic fusion proteins and selective delivery in the brain for the therapy of GBM.

Mouse skull is easily penetrated by diagnostic ultrasound at MHz frequencies. Therefore, clinical ultrasound imaging can monitor microbubbles in the murine brain vasculature in real time. While nonspecific binding of microbubbles onto vessel wall is low, molecular targeting can be applied to achieve selective adhesion to the vascular biomarkers of disease, such as markers of endothelial inflammation or tumor neovasculature, enabling contrast-specific imaging. Super-resolution imaging of ultrasound contrast microbubbles implies the computation of particle centroids, which improves spatial resolution of contrast ultrasound well beyond the dimensions of the point-spread function on the scanner screen, and the wavelength of ultrasound. Rapid visualization of moving and adherent bubbles, determination of velocity of the particles in blood flow, and distinguishing targeted bubbles becomes possible. With the advancement of all these approaches, undesired bioeffects of the combination of intravascular microbubbles and ultrasound must be noted. It has been observed that the use of clinical imaging equipment, with clinical imaging transducers, in combination of circulating microbubbles, may induce significant bioeffects, such as blood brain barrier opening, with potential for drug delivery, or unwanted bioeffects. Overall, the use of microbubble contrast agents provides quick and inexpensive tool for the assessment of brain vasculature in small experimental animals.

Primary, glioblastoma, and secondary brain tumors, from metastases outside the brain, are among the most aggressive and therapeutically resistant cancers. A physiological barrier protecting the brain, the blood-brain barrier (BBB), functions as a deterrent to effective therapies. To enhance cancer therapy, we developed a cancer terminator virus (CTV), a unique tropism-modified adenovirus consisting of serotype 3 fiber knob on an otherwise Ad5 capsid that replicates in a cancer-selective manner and simultaneously produces a potent therapeutic cytokine, melanoma differentiation-associated gene-7/interleukin-24 (MDA-7/IL-24). A limitation of the CTV and most other viruses, including adenoviruses, is an inability to deliver systemically to treat brain tumors because of the BBB, nonspecific virus trapping, and immune clearance. These obstacles to effective viral therapy of brain cancer have now been overcome using focused ultrasound with a dual microbubble treatment, the focused ultrasound-double microbubble (FUS-DMB) approach. Proof-of-principle is now provided indicating that the BBB can be safely and transiently opened, and the CTV can then be administered in a second set of complement-treated microbubbles and released in the brain using focused ultrasound. Moreover, the FUS-DMB can be used to deliver the CTV multiple times in animals with glioblastoma  growing in their brain thereby resulting in a further enhancement in survival. This strategy permits efficient therapy of primary and secondary brain tumors enhancing animal survival without promoting harmful toxic or behavioral side effects. Additionally, when combined with a standard of care therapy, Temozolomide, a further increase in survival is achieved. The FUS-DMB approach with the CTV highlights a noninvasive strategy to treat brain cancers without surgery. This innovative delivery scheme combined with the therapeutic efficacy of the CTV provides a novel potential translational therapeutic approach for brain cancers.

Ultrasound-triggered microbubbles destruction leading to vascular shutdown have resulted in preclinical studies in tumor growth delay or inhibition, lesion formation, radio-sensitization and modulation of the immune micro-environment.Antivascular ultrasound aims to be developed as a focal, targeted, non-invasive, mechanical and non-thermal treatment, alone or in combination with other treatments, and this review positions these treatments among the wider therapeutic ultrasound domain.Antivascular effects have been reported for a wide range of ultrasound exposure conditions, and evidence points to a prominent role of cavitation as the main mechanism.At relatively low peak negative acoustic pressure, predominantly non-inertial cavitation is most likely induced, while higher peak negative pressures lead to inertial cavitation and bubbles collapse.Resulting bioeffects start with inflammation and/or loose opening of the endothelial lining of the vessel.The latter causes vascular access of tissue factor, leading to platelet aggregation, and consequent clotting.Alternatively, endothelium damage exposes subendothelial collagen layer, leading to rapid adhesion and aggregation of platelets and clotting.In a pilot clinical trial, a prevalence of tumor response was observed in patients receiving ultrasound-triggered microbubble destruction along with transarterial radioembolization.Two ongoing clinical trials are assessing the effectiveness of ultrasound-stimulated microbubble treatment to enhance radiation effects in cancer patients.Clinical translation of antivascular ultrasound/microbubble approach may thus be forthcoming.

Objectives The objective of this study is to validate the modulated acoustic radiation force (mARF)–based imaging method in the detection of abdominal aortic aneurysm (AAA) in murine models using vascular endothelial growth factor receptor 2 (VEGFR-2)–targeted microbubbles (MBs). Materials and Methods The mouse AAA model was prepared using the subcutaneous angiotensin II (Ang II) infusion combined with the β-aminopropionitrile monofumarate solution dissolved in drinking water. The ultrasound imaging session was performed at 7 days, 14 days, 21 days, and 28 days after the osmotic pump implantation. For each imaging session, 10 C57BL/6 mice were implanted with Ang II–filled osmotic pumps, and 5 C57BL/6 mice received saline infusion only as the control group. Biotinylated lipid MBs conjugated to either anti–mouse VEGFR-2 antibody (targeted MBs) or isotype control antibody (control MBs) were prepared before each imaging session and were injected into mice via tail vein catheter. Two separate transducers were colocalized to image the AAA and apply ARF to translate MBs simultaneously. After each imaging session, tissue was harvested and the aortas were used for VEGFR-2 immunostaining analysis. From the collected ultrasound image data, the signal magnitude response of the adherent targeted MBs was analyzed, and a parameter, residual-to-saturation ratio ( R res − sat ), was defined to measure the enhancement in the adherent targeted MBs signal after the cessation of ARF compared with the initial signal intensity. Statistical analysis was performed with the Welch t test and analysis of variance test. Results The R res − sat of abdominal aortic segments from Ang II–challenged mice was significantly higher compared with that in the saline-infused control group ( P < 0.001) at all 4 time points after osmotic pump implantation (1 week to 4 weeks). In control mice, the R res − sat values were 2.13%, 1.85%, 3.26%, and 4.85% at 1, 2, 3, and 4 weeks postimplantation, respectively. In stark contrast, the R res − sat values for the mice with Ang II–induced AAA lesions were 9.20%, 20.6%, 22.7%, and 31.8%, respectively. It is worth noting that there was a significant difference between the R res − sat for Ang II–infused mice at all 4 time points ( P < 0.005), a finding not present in the saline-infused mice. Immunostaining results revealed the VEGFR-2 expression was increased in the abdominal aortic segments of Ang II–infused mice compared with the control group. Conclusions The mARF-based imaging technique was validated in vivo using a murine model of AAA and VEGFR-2–targeted MBs. Results in this study indicated that the mARF-based imaging technique has the ability to detect and assess AAA growth at early stages based on the signal intensity of adherent targeted MBs, which is correlated with the expression level of the desired molecular biomarker. The results may suggest, in very long term, a pathway toward eventual clinical implementation for an ultrasound molecular imaging–based approach to AAA risk assessment in asymptomatic patients.

Objective. This paper is an initial work towards developing particle-mediated histotripsy (PMH) as a novel method of treating catheter-based medical device (CBMD) intraluminal biofilms. Impact Statement. CBMDs commonly become infected with bacterial biofilms leading to medical device failure, infection, and adverse patient outcomes. Introduction. Histotripsy is a noninvasive focused ultrasound ablation method that was recently proposed as a novel method to remove intraluminal biofilms. Here, we explore the potential of combining histotripsy with acoustically active particles to develop a PMH approach that can noninvasively remove biofilms without the need for high acoustic pressures or real-time image guidance for targeting. Methods. Histotripsy cavitation thresholds in catheters containing either gas-filled microbubbles (MBs) or fluid-filled nanocones (NCs) were determined. The ability of these particles to sustain cavitation over multiple ultrasound pulses was tested after a series of histotripsy exposures. Next, the ability of PMH to generate selective intraluminal cavitation without generating extraluminal cavitation was tested. Finally, the biofilm ablation and bactericidal capabilities of PMH were tested using both MBs and NCs. Results. PMH significantly reduced the histotripsy cavitation threshold, allowing for selective luminal cavitation for both MBs and NCs. Results further showed PMH successfully removed intraluminal biofilms in Tygon catheters. Finally, results from bactericidal experiments showed minimal reduction in bacteria viability. Conclusion. The results of this study demonstrate the potential for PMH to provide a new modality for removing bacterial biofilms from CBMDs and suggest that additional work is warranted to develop histotripsy and PMH for treatment of CBMD intraluminal biofilms.

Mouse models are critical in developing new therapeutic approaches to treat peripheral arterial disease (PAD). Despite decades of research and numerous clinical trials, the efficacy of available therapies is limited. This may suggest shortcomings in our current animal models and/or methods of assessment. We evaluated perfusion measurement methods in a mouse model of PAD by comparing laser Doppler perfusion imaging (LDPI, the most common technique), contrast-enhanced ultrasound (CEUS, an emerging technique) and fluorescent microspheres (conventional standard). Mice undergoing a femoral artery ligation were assessed by LDPI and CEUS at baseline and 1, 4, 7, 14, 28, 60, 90 and 150 d post-surgery to evaluate perfusion recovery in the ischemic hindlimb. Fourteen days after surgery, additional mice were measured with fluorescent microspheres, LDPI, and CEUS. LDPI and CEUS resulted in broadly similar trends of perfusion recovery until 7 d post-surgery. However, by day 14, LDPI indicated full recovery of perfusion, whereas CEUS indicated ∼50% recovery, which failed to improve even after 5 mo. In agreement with the CEUS results, fluorescent microspheres at day 14 post-surgery confirmed that perfusion recovery was incomplete. Histopathology and photoacoustic microscopy provided further evidence of sustained vascular abnormalities.

Malignant mesothelioma (MM) is a lethal tumor originating in the mesothelium with high chemotherapeutic resistance. Cancer stem cells (CSCs) persist in tumors and are critical targets responsible for tumor resistance and recurrence. The identification and characterization of CSCs may help develop effective treatment for MM. The objective of this study was to evaluate the therapeutic effect of molecular targeted radiotherapy by 177Lu-labeled immunoliposomes (177Lu-ILs) on CSCs of mesothelioma. MM CSCs were sorted based on CD26/CD24 expression level and their functional significances were established by small interference RNA. CSC potential of MM was evaluated for drug resistance, cell invasion, and cell growth rate in vitro. CSC metabolism was evaluated with the uptake of 18F-FDG. Therapeutic effects of 177Lu-labeled immunoliposomes targeting CD26 and CD24 were evaluated in vitro through proliferation and apoptotic assays. CSCs sorted from H28 cells exhibited significant drug resistance and enhanced proliferative activity as well as increased metabolism indicated by higher 18F-FDG uptake. Treatment with 177Lu-ILs, compared with 177Lu-CL and ILs, showed enhanced therapeutic effects on inhibition of proliferation, up-regulation of apoptosis, and suppression of CD26 and CD24 expression. Thus, our results suggest that molecular radiotherapy targeting both CD26 and CD24 could be a promising approach for CSC-targeting therapy for MM.

Ultrasound molecular imaging techniques rely on the separation and identification of three types of signals: static tissue, adherent microbubbles and non-adherent microbubbles. In this study, the image filtering techniques of singular value thresholding (SVT) and normalized singular spectrum area (NSSA) were combined to isolate and identify vascular endothelial growth factor receptor 2-targeted microbubbles in a mouse hindlimb tumor model (n = 24). By use of a Verasonics Vantage 256 imaging system with an L12-5 transducer, a custom-programmed pulse inversion sequence employing synthetic aperture virtual source element imaging was used to collect contrast images of mouse tumors perfused with microbubbles. SVT was used to suppress static tissue signals by 9.6 dB while retaining adherent and non-adherent microbubble signals. NSSA was used to classify microbubble signals as adherent or non-adherent with high accuracy (receiver operating characteristic area under the curve [ROC AUC] = 0.97), matching the classification performance of differential targeted enhancement. The combined SVT + NSSA filtering method also outperformed differential targeted enhancement in differentiating MB signals from all other signals (ROC AUC = 0.89) without necessitating destruction of the contrast agent. The results from this study indicate that SVT and NSSA can be used to automatically segment and classify contrast signals. This filtering method with potential real-time capability could be used in future diagnostic settings to improve workflow and speed the clinical uptake of ultrasound molecular imaging techniques.

Objective: Acute hyperglycemia during myocardial infarction worsens outcomes in part by inflammatory mechanisms. Pulsed ultrasound has anti-inflammatory potential in bone healing and neuromodulation. We hypothesized that pulsed ultrasound would attenuate the hyperglycemic exacerbation of myocardial ischemia-reperfusion injury via the cholinergic anti-inflammatory pathway. Methods: Acute hyperglycemia was induced in wild-type C(57)BL6 or acetylcholine-receptor knockout (alpha 7nAChR(-/-)) mice by intraperitoneal injection of glucose. Pulsed ultrasound (frequency 7 MHz, bursting mechanical index 1.2, duration 1 second, repeated every 6 seconds for 2 minutes, 20-second total exposure) was performed at the spleen or neck after glucose injection. Separate mice underwent vagotomy before treatment. The left coronary artery was occluded for 20 minutes, followed by 60 minutes of reperfusion. The primary end point was infarct size in explanted hearts. Results: Splenic pulsed ultrasound significantly decreased infarct size in wild-type C57BL6 mice exposed to acute hyperglycemia and myocardial ischemia-reperfusion injury (5.2% +/- 4.4% vs 16.9% +/- 12.5% of risk region, P = .013). Knockout of alpha 7nAChR abrogated the beneficial effect of splenic pulsed ultrasound (22.2% +/- 12.1%, P = .79 vs control). Neck pulsed ultrasound attenuated the hyperglycemic exacerbation of myocardial infarct size (3.5% +/- 4.8%, P = .004 vs control); however, the cardioprotective effect disappeared in mice that underwent vagotomy. Plasma acetylcholine, beta(2) adrenergic receptor, and phosphorylated Akt levels were increased after splenic pulsed ultrasound treatment. Conclusions: Pulsed ultrasound treatment of the spleen or neck attenuated the hyperglycemic exacerbation of myocardial ischemia-reperfusion injury leading to a 3-fold decrease in infarct size. Pulsed ultrasound may provide cardioprotection via the cholinergic anti-inflammatory pathway and could be a promising new nonpharmacologic, noninvasive therapy to reduce infarct size during acute myocardial infarction and improve patient outcomes.

Introduction: Currently, the only disease-modifying therapy for degenerative mitral stenosis (DMS) due to mitral annular calcification (MAC) is open surgical valve repair or replacement, but it is technically challenging, and patients with DMS are often at elevated risk of surgical complications. As such, there is an unmet need for less-invasive strategies to treat DMS. Given the ability of shock-wave grade therapeutic ultrasound to disrupt calcifications, we set out to determine whether therapeutic ultrasound may be a possible treatment strategy for DMS. Hypothesis: This study aimed to evaluate whether ex vivo histotripsy can produce internal disruption of calcified nodules in the mitral annulus, a precursor to improving valve function in DMS. Methods: All experiments were performed on a formalin-fixed human cadaveric heart with severe MAC (Figure 1 A). Histotripsy cavitation was directed at calcified nodules utilizing a sonic concepts HIFUPlex Focused 128 element,1.1MHz array therapy transducer (Figure 1 E). B-mode ultrasound imaging was used to guide therapy. Transducer settings were PRF: 60 Hz, Duty cycle: 8x10^-4, transmit frequency: 1.1 MHz, HIFU Voltage: 60 V, resulting in an estimated pressure output of 34.6 MPa_pk. Results: A total of three histotripsy experiments were performed on the primary dominant calcium nodule. Visual assessment of treatment X-ray microtomography (Micro-CT) revealed qualitative fracturing of the calcified nodule from baseline (Figure 1 B), after the first treatment (Figure 1 C), and after three treatments (Figure 1 D). Gross visual inspection and Micro-CT demonstrated no changes to the surrounding fibrous annulus. Conclusions: In a feasibility study involving a fixed cadaveric heart, histotripsy therapeutic ultrasound demonstrated the potential to internally disrupt a MAC nodule without injuring the mitral annulus. Further studies are needed to address the ability of histotripsy to improve valve function in DMS.

Targeting of microbubbles (ultrasound contrast agents for molecular imaging) has been researched for more than two decades. However, methods of microbubble preparation and targeting ligand attachment are cumbersome, complicated, and lengthy. Therefore, there is a need to simplify the targeted microbubble preparation procedure to bring it closer to clinical translation. The purpose of this publication is to provide a detailed description and explanation of the steps necessary for targeted microbubble preparation, functional characterization and testing. A sequence of the optimized and simplified procedures is presented for two systems: a biotin-streptavidin targeting pair model, and a cyclic RGD peptide targeting the recombinant αvβ3 protein, which is overexpressed on the endothelial lining of the tumor neovasculature. Here, we show the following: covalent coupling of the targeting ligand to a lipid anchor, assessment of the reagent quality, and tests that confirm the successful completion of the reaction; preparation of the aqueous precursor medium containing microbubble shell components, followed by microbubble preparation via amalgamation; assessment of the efficacy of lipid transfer onto the microbubble stabilizer shell; adjustment of microbubble size distribution by flotation at normal gravity to remove larger microbubbles that might be detrimental for in vivo use; assessment of microbubble size distribution by electrozone sensing; evaluation of targeted binding of the microbubbles to receptor-coated surface in a static binding assay test (in an inverted dish); and evaluation of targeted binding of the microbubbles to receptor-coated surface in a parallel plate flow chamber test.

OBJECTIVE Surgery can be highly effective for the treatment of medically intractable, neurological disorders, such as drug-resistant focal epilepsy. However, despite its benefits, surgery remains substantially underutilized due to both surgical concerns and nonsurgical impediments. In this work, the authors characterized a noninvasive, nonablative strategy to focally destroy neurons in the brain parenchyma with the goal of limiting collateral damage to nontarget structures, such as axons of passage. METHODS Low-intensity MR-guided focused ultrasound (MRgFUS), together with intravenous microbubbles, was used to open the blood-brain barrier (BBB) in a transient and focal manner in rats. The period of BBB opening was exploited to focally deliver to the brain parenchyma a systemically administered neurotoxin (quinolinic acid) that is well tolerated peripherally and otherwise impermeable to the BBB. RESULTS Focal neuronal loss was observed in targeted areas of BBB opening, including brain regions that are prime objectives for epilepsy surgery. Notably, other structures in the area of neuronal loss, including axons of passage, glial cells, vasculature, and the ventricular wall, were spared with this procedure. CONCLUSIONS These findings identify a noninvasive, nonablative approach capable of disconnecting neural circuitry while limiting the neuropathological consequences that attend other surgical procedures. Moreover, this strategy allows conformal targeting, which could enhance the precision and expand the treatment envelope for treating irregularly shaped surgical objectives located in difficult-to-reach sites. Finally, if this strategy translates to the clinic, the noninvasive nature and specificity of the procedure could positively influence both physician referrals for and patient confidence in surgery for medically intractable neurological disorders.

Purpose: Chronic pancreatitis (CP) is an inflammatory disorder of the pancreas that leads to impaired pancreatic function. The limited therapeutic options and the lack of molecular targeting ligands or non-serum-based biomarkers hinder the development of target-specific drugs. Thus, there is a need for an unbiased, comprehensive discovery and evaluation of pancreatitis-specific ligands. Methods: This study utilized a computational-guided in vivo phage display approach to select peptide ligands selective for cellular components in the caerulein-induced mouse model of CP. The identified peptides were conjugated to pegylated DOPC liposomes via the reverse-phase evaporation method, and the in vivo specificity and pharmacokinetics were determined. As proof of concept, CP-targeted liposomes were used to deliver an antifibrotic small molecular drug, apigenin. Antifibrotic effects determined by pancreatic histology, fibronectin expression, and collagen deposition were evaluated. Results: We have identified five peptides specific for chronic pancreatitis and demonstrated selectivity to activated pancreatic stellate cells, acinar cells, macrophages, and extracellular matrix, respectively. MDLSLKP-conjugated liposomes demonstrated an increased particle accumulation by 1.3-fold in the inflamed pancreas compared to the control liposomes. We also observed that targeted delivery of apigenin resulted in improved acini preservation, a 37.2% and 33.1% respective reduction in collagen and fibronectin expression compared to mice receiving the free drug, and reduced oxidative stress in the liver. Conclusion: In summary, we have developed a systematic approach to profile peptide ligands selective for cellular components of complex disease models and demonstrated the biomedical applications of the identified peptides to improve tissue remodeling in the inflamed pancreas.