Modulating Nonlinear Acoustic Response of Phospholipid-Coated Microbubbles with pH for Ultrasound Imaging.

Activatable microbubble contrast agents for contrast-enhanced ultrasound have a potential role for measuring physiologic and pathologic states in deep tissues, including tumor acidosis. In this study, we describe a novel observation of increased harmonic oscillation of phosphatidylcholine microbubbles (PC-MBs) in response to lower ambient pH using a clinical ultrasound scanner. MB echogenicity and nonlinear echoes were monitored at neutral and acidic pH using B-mode and Cadence contrast pulse sequencing (CPS), a harmonic imaging technique at 7.0 and 1.5 MHz. A 3-fold increase in harmonic signal intensity was observed when the pH of PC-MB suspensions was decreased from 7.4 to 5.5 to mimic normal and pathophysiological levels that can be encountered in vivo. This pH-mediated activation is tunable based on the chemical structure and length of phospholipids composing the MB shell. It is also reliant on the presence of phosphate groups, as the use of lipids without phosphate instead of phospholipids completely abrogated this phenomenon. The increased harmonic signal likely is the result of increased MB oscillation caused by a decrease of the interfacial tension induced at a lower pH, altering the lipid conformation. While relative signal changes are interpreted clinically as mostly related to blood flow, pH effects could be significant contributors, particularly when imaging tumors. While our observation can be used clinically, it requires further research to isolate the effect of pH from other variables. These findings could pave the way toward for the development of new smart ultrasound contrast agents that expand the clinical utility of contrast-enhanced ultrasound.

In Vivo Ultrasound Imaging of Macrophages Using Acoustic Vaporization of Internalized Superheated Nanodroplets.

Activating patients' immune cells, either by reengineering them or treating them with bioactive molecules, has been a breakthrough in the field of immunotherapy and has revolutionized treatment, especially against cancer. As immune cells naturally home to tumors or injured tissues, labeling such cells holds promise for non-invasive tracking and biologic manipulation. Our study demonstrates that macrophages loaded with extremely low boiling point perfluorocarbon nanodroplets not only survive ultrasound-induced phase change but also maintain their phagocytic function. Unlike observations made when using higher boiling point perfluorocarbon nanodroplets, our results show that phase change occurs intracellularly at a low mechanical index using a clinical scanner operating within the energy limit set by the Food and Drug Administration (FDA). After nanodroplet-loaded macrophages were given intravenously to nude rats, they were invisible in the liver when imaged at a very low mechanical index using a clinical ultrasound scanner. They became visible when power was increased but still within the FDA limits up to 8 h after administration. The acoustic labeling and in vivo detection of macrophages using a clinical ultrasound scanner represent a paradigm shift in the field of cell tracking and pave the way for potential therapeutic strategies in the clinical setting.

Cancer immunotherapy based on image-guided STING activation by nucleotide nanocomplex-decorated ultrasound microbubbles.

The cytosolic innate immune sensor cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway is crucial for priming adaptive antitumour immunity through antigen-presenting cells (APCs). Natural agonists, such as cyclic dinucleotides (CDNs), activate the cGAS-STING pathway, but their clinical translation is impeded by poor cytosolic entry and serum stability, low specificity and rapid tissue clearance. Here we developed an ultrasound (US)-guided cancer immunotherapy platform using nanocomplexes composed of 2'3'-cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) electrostatically bound to biocompatible branched cationic biopolymers that are conjugated onto APC-targeting microbubbles (MBs). The nanocomplex-conjugated MBs engaged with APCs and efficiently delivered cGAMP into the cytosol via sonoporation, resulting in activation of cGAS-STING and downstream proinflammatory pathways that efficiently prime antigen-specific T cells. This bridging of innate and adaptive immunity inhibited tumour growth in both localized and metastatic murine cancer models. Our findings demonstrate that targeted local activation of STING in APCs under spatiotemporal US stimulation results in systemic antitumour immunity and improves the therapeutic efficacy of checkpoint blockade, thus paving the way towards novel image-guided strategies for targeted immunotherapy of cancer.

Catalase-Loaded Silica Nanoparticles Formulated via Direct Surface Modification as Potential Oxygen Generators for Hypoxia Relief.

Enzymes are biological catalysts that have many potential industrial and biomedical applications. However, the widespread use of enzymes in the industry has been limited by their instability and poor recovery. In biomedical applications, systemic administration of enzymes has faced two main challenges: limited bioactivity mostly due to rapid degradation by proteases and immunogenic activity, since most enzymes are from nonhuman sources. Herein, we propose a robust enzyme-encapsulation strategy to mitigate these limitations. Catalase (CAT) was encapsulated in nanoporous silica nanoparticles (CAT-SiNPs) by first chemically modifying the enzyme surface with a silica precursor, followed by silica growth and finally poly(ethylene glycol) (PEG) conjugation. The formulation was carried out in mild aqueous conditions and yielded nanoparticles (NPs) with a mean diameter of 230 ± 10 nm and a concentration of 1.3 ± 0.8 × 1012 NPs/mL. CAT-SiNPs demonstrated high enzyme activity, optimal protection from proteolysis by proteinase K and trypsin, and excellent stability over time. In addition, a new electrochemical assay was developed to measure CAT activity in a rapid, simple, and accurate manner without interference from chromophore usually present in biological samples. Concentrations of 2.5 × 1010 to 80 × 1010 CAT-SiNPs/mL not only proved to be nontoxic in cell cultures using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay but also conferred cell protection when cells were exposed to 1 mM hydrogen peroxide (H2O2). Finally, the ability of CAT-SiNPs to release oxygen (O2) when exposed to H2O2 was demonstrated in vivo using a rat model. Following the direct injection of CAT-SiNPs in the left kidney, partial pressure of oxygen (pO2) increased by more than 30 mmHg compared to the contralateral control kidney during the systemic infusion of safe levels of H2O2. This pilot study highlights the potential of CAT-SiNPs to generate O2 to relieve hypoxia in tissues and potentially sensitize tumors against radiation therapy.

Microbubbles Cloaked with Hydrogels as Activatable Ultrasound Contrast Agents.

Microbubbles (MBs) are optimal ultrasound contrast agents because their unique acoustic response allows for exquisite sensitivity in vivo. This unique response is derived from MBs' elasticity that allows them to oscillate differently from surrounding tissues. While the main use of MBs in the clinic is for cardiac and perfusion imaging, imparting MBs with bioresponsive properties would expand their use to detect pathophysiologic changes. This can be achieved by damping MBs' oscillations to silence their signal and rescuing it when they encounter the biomarker of interest to improve detection and specificity of diseases such as deep vein thrombosis (DVT). Here, we demonstrate that conjugating perfluorobutane-filled MBs with hyaluronic acid (HA) and cross-linking HA with biodegradable linkers eliminates harmonic signal because of increased MB stiffness and decreased oscillation. In this proof-of-concept study, we used a reversible pH-sensitive cross-linker to establish and validate this targeted and activatable pH-sensitive MB (pH-MB) platform. Conjugation of HA to MBs and targeting of pH-MBs to CD44-positive cells were validated. Harmonic signal loss due to stiffening of pH-MBs' shell was confirmed using a clinical ultrasound scanner equipped with Cadence contrast pulse sequencing. pH-MBs imaged before and after acidification increased harmonic signal fivefold. Because the cleavage of the cross-linker we used is reversible, harmonic signal was silenced again when the acidic suspension was neutralized, confirming that harmonic signal is dependent on the cross-linked HA. The rate of rise and the magnitude of harmonic signal increase could be manipulated by varying the phospholipid composition and the number of HA cross-linkers, indicating that the platform can be tuned to the desired response needed. In this study, we established the feasibility of using targeted and activatable MBs and plan to apply this platform to aid in the diagnosis and management of patients with DVT and potentially other conditions.

Bubble Inflation Using Phase-Change Perfluorocarbon Nanodroplets as a Strategy for Enhanced Ultrasound Imaging and Therapy.

Phase-change perfluorocarbon microdroplets were introduced over 2 decades ago to occlude downstream vessels in vivo. Interest in perfluorocarbon nanodroplets has recently increased to enable extravascular targeting, to rescue the weak ultrasound signal of perfluorocarbon droplets by converting them to microbubbles and to improve ultrasound-based therapy. Despite great scientific interest and advances, applications of phase-change perfluorocarbon agents have not reached clinical testing because of efficacy and safety concerns, some of which remain unexplained. Here, we report that the coexistence of perfluorocarbon droplets and microbubbles in blood, which is inevitable when droplets spontaneously or intentionally vaporize to form microbubbles, is a major contributor to the observed side effects. We develop the theory to explain why the coexistence of droplets and microbubbles results in microbubble inflation induced by perfluorocarbon transfer from droplets to adjacent microbubbles. We also present the experimental data showing up to 6 orders of magnitude microbubble volume expansion, which occludes a 200 µm tubing in the presence of perfluorocarbon nanodroplets. More importantly, we demonstrate that the rate of microbubble inflation and ultimate size can be controlled by manipulating formulation parameters to tailor the agent's design for the potential theranostic application while minimizing the risk to benefit ratio.

Fluorous-phase iron oxide nanoparticles as enhancers of acoustic droplet vaporization of perfluorocarbons with supra-physiologic boiling point.

Perfluorocarbon emulsion nanodroplets containing iron oxide nanoparticles (IONPs) within their inner perfluorohexane (PFH) core were prepared to investigate potential use as an acoustically activatable ultrasound contrast agent, with the hypothesis that incorporation of IONPs into the fluorous phase of a liquid perfluorocarbon emulsion would potentiate acoustic vaporization. IONPs with an oleic acid (OA) hydrophobic coating were synthesized through chemical co-precipitation. To suspend IONP in PFH, OA was exchanged with perfluorononanoic acid (PFNA) via ligand exchange to yield fluorophilic PFNA-coated IONPs (PFNA-IONPs). Suspensions with various amounts of PFNA-IONPs (0-15% w/v) in PFH were emulsified in saline by sonication, using 5% (w/v) egg yolk phospholipid as an emulsifier. PFNA-IONPs were characterized with transmission electron microscopy (TEM), transmission electron cryomicroscopy (cryoTEM), and thermogravimetric analysis (TGA) with Fourier transform infrared spectroscopy (FTIR). IONP were between 5 and 10 nm in diameter as measured by electron microscopy, and hydrodynamic size of the PFH nanodroplets were 150 to 230 nm as measured by dynamic light scattering (DLS). Acoustic droplet vaporization of PFH nanodroplets (PFH-NDs) was induced using conversion pulses (100 cycle at 1.1 MHz and 50% duty cycle) provided by a focused ultrasound transducer, and formed microbubbles were imaged using a clinical ultrasound scanner. The acoustic pressure threshold needed for PFH-NDs vaporization decreased with increasing temperature and IONP content. PFH-NDs containing 5% w/v IONP converted to microbubbles at 42 °C at 2.18 MI, which is just above the exposure limits of 1.9 MI allowed by the FDA for clinical ultrasound scanners, whereas 10 and 15% emulsion vaporized at 1.87 and 1.24 MI, respectively. Furthermore, 5% IONP-loaded PFH-NDs injected intravenously into melanoma-bearing mice at a dose of 120 mg PFH/kg, converted into detectable microbubbles in vivo 5 h, but not shortly after injection, indicating that this technique detects NDs accumulated in tumors.

Catalase-Containing Silica Particles as Ultrasound-Based Hydrogen Peroxide Sensors to Determine Infected From Noninfected Fluid Collections in Humans.

OBJECTIVE. Hydrogen peroxide (H2O2) plays a key role in neutrophil oxidative defense against infection. Catalase-containing silica nanoshells are nanoparticles that generate O2 microbubbles imaged with ultrasound in the presence of elevated H2O2. We aimed to determine whether ultrasound-detectable O2 microbubbles produced by catalase-containing silica nanoshells can determine whether fluid collections drained from patients are infected. SUBJECTS AND METHODS. During this HIPAA-compliant, institutional review board-approved study, 52 human fluid samples were collected from clinically required image-guided percutaneous drainage procedures. Catalase-containing silica nanoshells were added to the fluid samples during imaging in real time using a Sequoia-512 15L8-S linear transducer (Siemens Healthcare). Production of detectable microbubbles was graded subjectively as negative (noninfected) or positive (infected) with low, moderate, or high confidence by a single observer blinded to all clinical data. The truth standard was microbiology laboratory culture results. Performance characteristics including ROC curves were calculated. RESULTS. Microbubble detection to distinguish infected from noninfected fluids was 84% sensitive and 72% specific and offered negative and positive predictive values of 89% and 64%, respectively. The AUC was 0.79. Six of nine false-positive samples were peritoneal fluid collections that were all collected from patients with decompensated cirrhosis. CONCLUSION. The presence of elevated H2O2 indicated by microbubble formation in the presence of catalase-containing silica nanoshells is sensitive in distinguishing infected from noninfected fluids and offers a relatively high negative predictive value. False-positive cases may result from noninfectious oxidative stress. Catalase-containing silica nanoshells may constitute a novel point-of-care test performed at time of percutaneous drainage, potentially obviating placement of drains into otherwise sterile collections and minimizing risk of secondary infection or other complication.

Advances in gadolinium-based MRI contrast agent designs for monitoring biological processes in vivo.

The gadolinium-based contrast agents widely used in diagnostic MRI exams for 30 years are all small molecule agents that distribute into all extracellular spaces in tissues without providing any specific biological information. Although many 'responsive agent' designs have been presented over the past 20 years or so, none have found use in clinical diagnostic medicine at this point. This review summarizes some recent approaches taken to enhance the sensitivity of such gadolinium-based agents, to target them to specific tissue components, and to create new systems for monitoring specific biological processes.

Thrombin-Activatable Microbubbles as Potential Ultrasound Contrast Agents for the Detection of Acute Thrombosis.

Acute deep vein thrombosis (DVT) is the formation of a blood clot in the deep veins of the body that can lead to fatal pulmonary embolism. Acute DVT is difficult to distinguish from chronic DVT by ultrasound (US), the imaging modality of choice, and is therefore treated aggressively with anticoagulants, which can lead to internal bleeding. Here we demonstrate that conjugating perfluorobutane-filled (PFB-filled) microbubbles (MBs) with thrombin-sensitive activatable cell-penetrating peptides (ACPPs) could lead to the development of contrast agents that detect acute thrombosis with US imaging. Successful conjugation of ACPP to PFB-filled MBs was confirmed by fluorescence microscopy and flow cytometry. Fluorescein-labeled ACPP was used to evaluate the efficiency of thrombin-triggered cleavage by measuring the mean fluorescence intensity of ACPP-labeled MBs (ACPP-MBs) before and after incubation at 37 °C with thrombin. Lastly, control MBs and ACPP-MBs were infused through a tube containing a clot, and US contrast enhancement was measured with or without the presence of a thrombin inhibitor after washing the clot with saline. With thrombin activity, 91.7 ± 14.2% of the signal was retained after ACPP-MB infusion and washing, whereas only 16.7 ± 4% of the signal was retained when infusing ACPP-MBs in the presence of hirudin, a potent thrombin inhibitor.

Distinct ON/OFF fluorescence signals from dual-responsive activatable nanoprobes allows detection of inflammation with improved contrast.

Visualization of biochemical changes associated with disease is of great clinical significance, as it should allow earlier, more accurate diagnosis than structural imaging, facilitating timely clinical intervention. Herein, we report combining stimuli-responsive polymers and near-infrared fluorescent dyes (emission max: 790 nm) to create robust activatable fluorescent nanoprobes capable of simultaneously detecting acidosis and oxidative stress associated with inflammatory microenvironments. The spectrally-resolved mechanism of fluorescence activation allows removal of unwanted background signal (up to 20-fold reduction) and isolation of a pure activated signal, which enables sensitive and unambiguous localization of inflamed areas; target-to-background ratios reach 22 as early as 3 h post-injection. This new detection platform could have significant clinical impact in early detection of pathologies, individual tailoring of drug therapy, and image-guided tumor resection.

Layered hydrogels accelerate iPSC-derived neuronal maturation and reveal migration defects caused by MeCP2 dysfunction.

Probing a wide range of cellular phenotypes in neurodevelopmental disorders using patient-derived neural progenitor cells (NPCs) can be facilitated by 3D assays, as 2D systems cannot entirely recapitulate the arrangement of cells in the brain. Here, we developed a previously unidentified 3D migration and differentiation assay in layered hydrogels to examine how these processes are affected in neurodevelopmental disorders, such as Rett syndrome. Our soft 3D system mimics the brain environment and accelerates maturation of neurons from human induced pluripotent stem cell (iPSC)-derived NPCs, yielding electrophysiologically active neurons within just 3 wk. Using this platform, we revealed a genotype-specific effect of methyl-CpG-binding protein-2 (MeCP2) dysfunction on iPSC-derived neuronal migration and maturation (reduced neurite outgrowth and fewer synapses) in 3D layered hydrogels. Thus, this 3D system expands the range of neural phenotypes that can be studied in vitro to include those influenced by physical and mechanical stimuli or requiring specific arrangements of multiple cell types.

Light-triggered chemical amplification to accelerate degradation and release from polymeric particles.

We describe a means of chemical amplification to accelerate triggered degradation of a polymer and particles composed thereof. We designed a light-degradable copolymer containing carboxylic acids masked by photolabile groups and ketals. Photolysis allows the unmasked acidic groups in the polymer backbone to accelerate ketal hydrolysis even at neutral pH.

Short Soluble Coumarin Crosslinkers for Light-Controlled Release of Cells and Proteins from Hydrogels.

Materials that degrade or dissociate in response to low power light promise to enable on-demand, precisely localized delivery of drugs or bioactive molecules in living systems. Such applications remain elusive because few materials respond to wavelengths that appreciably penetrate tissues. The photocage bromohydroxycoumarin (Bhc) is efficiently cleaved upon low-power ultraviolet (UV) and near-infrared (NIR) irradiation through one- or two-photon excitation, respectively. We have designed and synthesized a short Bhc-bearing crosslinker to create light-degradable hydrogels and nanogels. Our crosslinker breaks by intramolecular cyclization in a manner inspired by the naturally occurring ornithine lactamization, in response to UV and NIR light, enabling rapid degradation of polyacrylamide gels and release of small hydrophilic payloads such as an ∼10 nm model protein and murine mesenchymal stem cells, with no background leakage.

Long-Lasting and Efficient Tumor Imaging Using a High Relaxivity Polysaccharide Nanogel Magnetic Resonance Imaging Contrast Agent.

Clinically approved small-molecule magnetic resonance imaging (MRI) contrast agents are all rapidly cleared from the body and offer weak signal enhancement. To avoid repeated administration of contrast agent and improve signal-to-noise ratios, agents with stronger signal enhancement and better retention in tumors are needed. Therefore, we focused on hydrogels because of their excellent water accessibility and biodegradability. Gadolinium (Gd)-chelating cross-linkers were incorporated into self-assembled pullulan nanogels to both impart magnetic properties and to stabilize this material that has been extensively studied for medical applications. We show that these Gd-chelating pullulan nanogels (Gd-CHPOA) have the highest reported relaxivity for any hydrogel-based particles and accumulate in the 4T1 tumors in mice at high levels 4 h after injection. This combination offers high signal enhancement and lasts up to 7 days to delineate the tumor clearly for longer imaging time scales. Importantly, this long-term accumulation does not cause any damage or toxicity in major organs up to three months after injection. Our work highlights the clinical potential of Gd-CHPOA as a tumor-imaging MRI contrast agent, permitting tumor identification and assessment with a high signal-to-background ratio.

Light-responsive nanoparticle depot to control release of a small molecule angiogenesis inhibitor in the posterior segment of the eye.

Therapies for macular degeneration and diabetic retinopathy require intravitreal injections every 4-8 weeks. Injections are uncomfortable, time-consuming, and carry risks of infection and retinal damage. However, drug delivery via noninvasive methods to the posterior segment of the eye has been a major challenge due to the eye's unique anatomy and physiology. Here we present a novel nanoparticle depot platform for on-demand drug delivery using a far ultraviolet (UV) light-degradable polymer, which allows noninvasively triggered drug release using brief, low-power light exposure. Nanoparticles stably retain encapsulated molecules in the vitreous, and can release cargo in response to UV exposure up to 30 weeks post-injection. Light-triggered release of nintedanib (BIBF 1120), a small molecule angiogenesis inhibitor, 10 weeks post-injection suppresses choroidal neovascularization (CNV) in rats. Light-sensitive nanoparticles are biocompatible and cause no adverse effects on the eye as assessed by electroretinograms (ERG), corneal and retinal tomography, and histology.

Nanogels from metal-chelating crosslinkers as versatile platforms applied to copper-64 PET imaging of tumors and metastases.

Metals are essential in medicine for both therapy and diagnosis. We recently created the first metal-chelating nanogel imaging agent, which employed versatile, reproducible chemistry that maximizes chelation stability. Here we demonstrate that our metal chelating crosslinked nanogel technology is a powerful platform by incorporating (64)Cu to obtain PET radiotracers. Polyacrylamide-based nanogels were crosslinked with three different polydentate ligands (DTPA, DOTA, NOTA). NOTA-based nanogels stably retained (64)Cu in mouse serum and accumulated in tumors in vivo as detected by PET/CT imaging. Measurement of radioactivity in major organs ex vivo confirmed this pattern, revealing a high accumulation (12.3% ID/g and 16.6% ID/g) in tumors at 24 and 48 h following administration, with lower accumulation in the liver (8.5% ID/g at 24 h) and spleen (5.5% ID/g). Nanogels accumulated even more efficiently in metastases (29.9% and 30.4% ID/g at 24 and 48 h). These metal-chelating nanogels hold great promise for future application as bimodal PET/MRI agents; chelation of ß-emitting radionuclides could enable radiation therapy.

Near-infrared-induced heating of confined water in polymeric particles for efficient payload release.

Near-infrared (NIR) light-triggered release from polymeric capsules could make a major impact on biological research by enabling remote and spatiotemporal control over the release of encapsulated cargo. The few existing mechanisms for NIR-triggered release have not been widely applied because they require custom synthesis of designer polymers, high-powered lasers to drive inefficient two-photon processes, and/or coencapsulation of bulky inorganic particles. In search of a simpler mechanism, we found that exposure to laser light resonant with the vibrational absorption of water (980 nm) in the NIR region can induce release of payloads encapsulated in particles made from inherently non-photo-responsive polymers. We hypothesize that confined water pockets present in hydrated polymer particles absorb electromagnetic energy and transfer it to the polymer matrix, inducing a thermal phase change. In this study, we show that this simple and highly universal strategy enables instantaneous and controlled release of payloads in aqueous environments as well as in living cells using both pulsed and continuous wavelength lasers without significant heating of the surrounding aqueous solution.

Synthesis and recognition studies with a ditopic, photoswitchable deep cavitand.

We describe the synthesis and photochemical behavior of open-ended container modules connected by a 4,4'-azobiphenyl spacer. Both trans and cis azo configurations of the host can be accessed and their binding of guest molecules was characterized by NMR methods.

Reversible switching between self-assembled homomeric and hybrid capsules.

We present here the reversible, guest-controlled disproportionation of homomeric and hybrid capsules using photochemistry. The supramolecular containers are self-assembled from shallow and deep cavitand modules.