Feature Extraction of Ultrasound Radiofrequency Data for the Classification of the Peripheral Zone of Human Prostate.

Prostate cancer ranks among the most prevalent types of cancer in males, prompting a demand for early detection and noninvasive diagnostic techniques. This paper explores the potential of ultrasound radiofrequency (RF) data to study different anatomic zones of the prostate. The study leverages RF data's capacity to capture nuanced acoustic information from clinical transducers. The research focuses on the peripheral zone due to its high susceptibility to cancer. The feasibility of utilizing RF data for classification is evaluated using ex-vivo whole prostate specimens from human patients. Ultrasound data, acquired using a phased array transducer, is processed, and correlated with B-mode images. A range filter is applied to highlight the peripheral zone's distinct features, observed in both RF data and 3D plots. Radiomic features were extracted from RF data to enhance tissue characterization and segmentation. The study demonstrated RF data's ability to differentiate tissue structures and emphasizes its potential for prostate tissue classification, addressing the current limitations of ultrasound imaging for prostate management. These findings advocate for the integration of RF data into ultrasound diagnostics, potentially transforming prostate cancer diagnosis and management in the future.

Remote Loading: The Missing Piece for Achieving High Drug Payload and Rapid Release in Polymeric Microbubbles.

The use of drug-loaded microbubbles for targeted drug delivery, particularly in cancer treatment, has been extensively studied in recent years. However, the loading capacity of microbubbles has been limited due to their surface area. Typically, drug molecules are loaded on or within the shell, or drug-loaded nanoparticles are coated on the surfaces of microbubbles. To address this significant limitation, we have introduced a novel approach. For the first time, we employed a transmembrane ammonium sulfate and pH gradient to load doxorubicin in a crystallized form in the core of polymeric microcapsules. Subsequently, we created remotely loaded microbubbles (RLMBs) through the sublimation of the liquid core of the microcapsules. Remotely loaded microcapsules exhibited an 18-fold increase in drug payload compared with physically loaded microcapsules. Furthermore, we investigated the drug release of RLMBs when exposed to an ultrasound field. After 120 s, an impressive 82.4 ± 5.5% of the loaded doxorubicin was released, demonstrating the remarkable capability of remotely loaded microbubbles for on-demand drug release. This study is the first to report such microbubbles that enable rapid drug release from the core. This innovative technique holds great promise in enhancing drug loading capacity and advancing targeted drug delivery.

H.O.S.T.: Hemoglobin microbubble-based Oxidative stress Sensing Technology.

In this work, we discuss the development of H.O.S.T., a novel hemoglobin microbubble-based electrochemical biosensor for label-free detection of Hydrogen peroxide (H2O2) towards oxidative stress and cancer diagnostic applications. The novelty of the constructed sensor lies in the use of a sonochemically prepared hemoglobin microbubble capture probe, which allowed for an extended dynamic range, lower detection limit, and enhanced resolution compared to the native hemoglobin based H2O2 biosensors. The size of the prepared particles Hemoglobin microbubbles was characterized using Coulter Counter analysis and was found to be 4.4 microns, and the morphology of these spherical microbubbles was shown using Brightfield microscopy. The binding chemistry of the sensor stack elements of HbMbs' and P.A.N.H.S. crosslinker was characterized using Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy and UV-Vis Spectroscopy. The electrochemical biosensor calibration (R2 > 0.95) was done using Electrochemical Impedance Spectroscopy, Cyclic Voltammetry, and Square Wave Voltammetry. The electrochemical biosensor calibration (R2 > 0.95) was done using Electrochemical Impedance Spectroscopy, Cyclic Voltammetry, and Square Wave Voltammetry. The specificity of the sensor for H2O2 was analyzed using cross-reactivity studies using ascorbic acid and glucose as interferents (p < 0.0001 for the highest non-specific dose versus the lowest specific dose). The developed sensor showed good agreement in performance with a commercially available kit for H2O2 detection using Bland Altman Analysis (mean bias = 0.37 for E.I.S. and - 24.26 for CV). The diagnostic potential of the biosensor was further tested in cancerous (N.G.P.) and non-cancerous (H.E.K.) cell lysate for H2O2 detection (p = 0.0064 for E.I.S. and p = 0.0062 for CV). The Michaelis Menten constant calculated from the linear portion of the sensor was found to be [Formula: see text] of 19.44 µM indicating that our biosensor has a higher affinity to Hydrogen peroxide than other available enzymatic sensors, it is attributed to the unique design of the hemoglobin polymers in microbubble.

Endothelial ERα promotes glucose tolerance by enhancing endothelial insulin transport to skeletal muscle.

The estrogen receptor (ER) designated ERα has actions in many cell and tissue types that impact glucose homeostasis. It is unknown if these include mechanisms in endothelial cells, which have the potential to influence relative obesity, and processes in adipose tissue and skeletal muscle that impact glucose control. Here we show that independent of impact on events in adipose tissue, endothelial ERα promotes glucose tolerance by enhancing endothelial insulin transport to skeletal muscle. Endothelial ERα-deficient male mice are glucose intolerant and insulin resistant, and in females the antidiabetogenic actions of estradiol (E2) are absent. The glucose dysregulation is due to impaired skeletal muscle glucose disposal that results from attenuated muscle insulin delivery. Endothelial ERα activation stimulates insulin transcytosis by skeletal muscle microvascular endothelial cells. Mechanistically this involves nuclear ERα-dependent upregulation of vesicular trafficking regulator sorting nexin 5 (SNX5) expression, and PI3 kinase activation that drives plasma membrane recruitment of SNX5. Thus, coupled nuclear and non-nuclear actions of ERα promote endothelial insulin transport to skeletal muscle to foster normal glucose homeostasis.

The Evolution and Recent Trends in Acoustic Targeting of Encapsulated Drugs to Solid Tumors: Strategies beyond Sonoporation.

Despite recent advancements in ultrasound-mediated drug delivery and the remarkable success observed in pre-clinical studies, no delivery platform utilizing ultrasound contrast agents has yet received FDA approval. The sonoporation effect was a game-changing discovery with a promising future in clinical settings. Various clinical trials are underway to assess sonoporation's efficacy in treating solid tumors; however, there are disagreements on its applicability to the broader population due to long-term safety issues. In this review, we first discuss how acoustic targeting of drugs gained importance in cancer pharmaceutics. Then, we discuss ultrasound-targeting strategies that have been less explored yet hold a promising future. We aim to shed light on recent innovations in ultrasound-based drug delivery including newer designs of ultrasound-sensitive particles specifically tailored for pharmaceutical usage.

Non-viral nitric oxide-based gene therapy improves perfusion and liposomal doxorubicin sonopermeation in neuroblastoma models.

Neuroblastoma (NB) is a pediatric malignancy that accounts for 15% of cancer-related childhood mortality. High-risk NB requires an aggressive chemoradiotherapy regimen that causes significant off-target toxicity. Despite this invasive treatment, many patients either relapse or do not respond adequately. Recent studies suggest that improving tumor perfusion can enhance drug accumulation and distribution within the tumor tissue, potentially augmenting treatment effects without inflicting systemic toxicity. Accordingly, methods that transiently increase tumor perfusion prior to treatment may help combat this disease. Here, we show the use of gene therapy to confer inducible nitric oxide synthase (iNOS) expression solely in the tumor space, using focused ultrasound targeting. NOS catalyzes the reaction that generates nitric oxide (NO), a potent endogenous vasodilator. This study reports the development of a targeted non-viral image-guided platform to deliver iNOS-expressing plasmid DNA (pDNA) to vascular endothelial cells encasing tumor blood vessels. Following transfection, longitudinal quantitative contrast-enhanced ultrasound (qCEUS) imaging revealed an increase in tumor perfusion over 72 h, attributed to elevated intratumoral iNOS expression. Methods: To construct a gene delivery vector, cationic ultrasound-responsive agents (known as "microbubbles") were employed to carry pDNA in circulation and transfect tumor vascular endothelial cells in vivo using focused ultrasound (FUS) energy. This was followed by liposomal doxorubicin (L-DOX) treatment. The post-transfection tumor response was monitored longitudinally using qCEUS imaging to determine relative changes in blood volumes and perfusion rates. After therapy, ex vivo analysis of tumors was performed to examine the bioeffects associated with iNOS expression. Results: By combining FUS therapy with cationic ultrasound contrast agents (UCAs), we achieved selective intratumoral transfection of pDNA encoding the iNOS enzyme. While transitory, the degree of expression was sufficient to induce significant increases in tumoral perfusion, to appreciably enhance the chemotherapeutic payload and to extend survival time in an orthotopic xenograft model. Conclusion: We have demonstrated the ability of a novel targeted non-viral gene therapy strategy to enhance tumor perfusion and improve L-DOX delivery to NB xenografts. While our results demonstrate that transiently increasing tumor perfusion improves liposome-encapsulated chemotherapeutic uptake and distribution, we expect that our iNOS gene delivery paradigm can also significantly improve radio and immunotherapies by increasing the delivery of radiosensitizers and immunomodulators, potentially improving upon current NB treatment without concomitant adverse effects. Our findings further suggest that qCEUS imaging can effectively monitor changes in tumor perfusion in vivo, allowing the identification of an ideal time-point to administer therapy.

Hemoglobin Microbubbles and the Prediction of Different Oxygen Levels Using RF Data and Deep Learning.

Ultrasound contrast agents (UCA) are gas-encapsulated microspheres that oscillate volumetrically when exposed to an ultrasound field producing backscattered signals efficiently, which can be used for improved ultrasound imaging and drug delivery applications. We developed a novel oxygen-sensitive hemoglobin-shell microbubble designed to acoustically detect blood oxygen levels. We hypothesize that structural change in hemoglobin caused due to varying oxygen levels in the body can lead to mechanical changes in the shell of the UCA. This can produce detectable changes in the acoustic response that can be used for measuring oxygen levels in the body. In this study, we have shown that oxygenated hemoglobin microbubbles can be differentiated from deoxygenated hemoglobin microbubbles using a 1D convolutional neural network using radiofrequency (RF) data. We were able to classify RF data from oxygenated and deoxygenated hemoglobin microbubbles into the two classes with a testing accuracy of 90.15%. The results suggest that oxygen content in hemoglobin affects the acoustical response and may be used for determining oxygen levels and thus could open many applications, including evaluating hypoxic regions in tumors and the brain, among other blood-oxygen-level-dependent imaging applications.

Remote Loading of Gas Bubbles into Polylactic Acid Microcapsules Creates Acoustically Active Janus Particles.

Polymeric microcapsules (MCs) are biocompatible agents used in biomedical applications such as drug delivery and in vivo imaging. We have discovered a method of remotely loading air into polylactic acid (PLA)-based MCs with an aqueous core. When the microcapsules are suspended in high content glycerol and propylene glycol solutions, changes in gas solubility cause bubbles to nucleate within the core through an "Ouzo-like" effect. The resulting bubble displaces the internal fluid of the MCs, but small molecules are retained in their interior. The residual content does not homogeneously distribute; rather, it localizes to one specific location, creating gas-filled Janus particles.

Improving Release of Liposome-Encapsulated Drugs with Focused Ultrasound and Vaporizable Droplet-Liposome Nanoclusters.

Active targeted delivery of small molecule drugs is becoming increasingly important in personalized therapies, especially in cancer, brain disorders, and a wide variety of other diseases. However, effective means of spatial targeting and delivering high drug payloads in vivo are still lacking. Focused ultrasound combined with superheated phase-shift nanodroplets, which vaporize into microbubbles using heat and sound, are rapidly becoming a popular strategy for targeted drug delivery. Focused ultrasound can target deep tissue with excellent spatial precision and without using ionizing energy, thus can activate nanodroplets in circulation. One of the main limitations of this technology has been poor drug loading in the droplet core or the shell material. To address this need, we have developed a strategy to combine low-boiling point decafluorabutane and octafluoropropane (DFB and OFP) nanodroplets with drug-loaded liposomes, creating phase-changeable droplet-liposome clusters (PDLCs). We demonstrate a facile method of assembling submicron PDLCs with high drug-loading capacity on the droplet surface. Furthermore, we demonstrate that chemical tethering of liposomes in PDLCs enables a rapid release of their encapsulated cargo upon acoustic activation (>60% using OFP-based PDLCs). Rapid uncaging of small molecule drugs would make them immediately bioavailable in target tissue or promote better penetration in local tissue following intravascular release. PDLCs developed in this study can be used to deliver a wide variety of liposome-encapsulated therapeutics or imaging agents for multi-modal imaging applications. We also outline a strategy to deliver a surrogate encapsulated drug, fluorescein, to tumors in vivo using focused ultrasound energy and PDLCs.

Non-invasive molecularly-specific millimeter-resolution manipulation of brain circuits by ultrasound-mediated aggregation and uncaging of drug carriers.

Non-invasive, molecularly-specific, focal modulation of brain circuits with low off-target effects can lead to breakthroughs in treatments of brain disorders. We systemically inject engineered ultrasound-controllable drug carriers and subsequently apply a novel two-component Aggregation and Uncaging Focused Ultrasound Sequence (AU-FUS) at the desired targets inside the brain. The first sequence aggregates drug carriers with millimeter-precision by orders of magnitude. The second sequence uncages the carrier's cargo locally to achieve high target specificity without compromising the blood-brain barrier (BBB). Upon release from the carriers, drugs locally cross the intact BBB. We show circuit-specific manipulation of sensory signaling in motor cortex in rats by locally concentrating and releasing a GABAA receptor agonist from ultrasound-controlled carriers. Our approach uses orders of magnitude (1300x) less drug than is otherwise required by systemic injection and requires very low ultrasound pressures (20-fold below FDA safety limits for diagnostic imaging). We show that the BBB remains intact using passive cavitation detection (PCD), MRI-contrast agents and, importantly, also by sensitive fluorescent dye extravasation and immunohistochemistry.

Perfusion-guided sonopermeation of neuroblastoma: a novel strategy for monitoring and predicting liposomal doxorubicin uptake in vivo.

Neuroblastoma (NB) is the most common extracranial solid tumor in infants and children, and imposes significant morbidity and mortality in this population. The aggressive chemoradiotherapy required to treat high-risk NB results in survival of less than 50%, yet is associated with significant long-term adverse effects in survivors. Boosting efficacy and reducing morbidity are therefore key goals of treatment for affected children. We hypothesize that these may be achieved by developing strategies that both focus and limit toxic therapies to the region of the tumor. One such strategy is the use of targeted image-guided drug delivery (IGDD), which is growing in popularity in personalized therapy to simultaneously improve on-target drug deposition and assess drug pharmacodynamics in individual patients. IGDD strategies can utilize a variety of imaging modalities and methods of actively targeting pharmaceutical drugs, however in vivo imaging in combination with focused ultrasound is one of the most promising approaches already being deployed for clinical applications. Over the last two decades, IGDD using focused ultrasound with "microbubble" ultrasound contrast agents (UCAs) has been increasingly explored as a method of targeting a wide variety of diseases, including cancer. This technique, known as sonopermeation, mechanically augments vascular permeability, enabling increased penetration of drugs into target tissue. However, to date, methods of monitoring the vascular bioeffects of sonopermeation in vivo are lacking. UCAs are excellent vascular probes in contrast-enhanced ultrasound (CEUS) imaging, and are thus uniquely suited for monitoring the effects of sonopermeation in tumors. Methods: To monitor the therapeutic efficacy of sonopermeation in vivo, we developed a novel system using 2D and 3D quantitative contrast-enhanced ultrasound imaging (qCEUS). 3D tumor volume and contrast enhancement was used to evaluate changes in blood volume during sonopermeation. 2D qCEUS-derived time-intensity curves (TICs) were used to assess reperfusion rates following sonopermeation therapy. Intratumoral doxorubicin (and liposome) uptake in NB was evalauted ex vivo along with associated vascular changes. Results: In this study, we demonstrate that combining focused ultrasound therapy with UCAs can significantly enhance chemotherapeutic payload to NB in an orthotopic xenograft model, by improving delivery and tumoral uptake of long-circulating liposomal doxorubicin (L-DOX) nanoparticles. qCEUS imaging suggests that changes in flow rates are highly sensitive to sonopermeation and could be used to monitor the efficacy of treatment in vivo. Additionally, initial tumor perfusion may be a good predictor of drug uptake during sonopermeation. Following sonopermeation treatment, vascular biomarkers show increased permeability due to reduced pericyte coverage and rapid onset of doxorubicin-induced apoptosis of NB cells but without damage to blood vessels. Conclusion: Our results suggest that significant L-DOX uptake can occur by increasing tumor vascular permeability with microbubble sonopermeation without otherwise damaging the vasculature, as confirmed by in vivo qCEUS imaging and ex vivo analysis. The use of qCEUS imaging to monitor sonopermeation efficiency and predict drug uptake could potentially provide real-time feedback to clinicians for determining treatment efficacy in tumors, leading to better and more efficient personalized therapies. Finally, we demonstrate how the IGDD strategy outlined in this study could be implemented in human patients using a single case study.

Impact of hydrostatic pressure on phase-change contrast agent activation by pulsed ultrasound.

A phase-change contrast agent (PCCA) can be activated from a liquid (nanodroplet) state using pulsed ultrasound (US) energy to form a larger highly echogenic microbubble (MB). PCCA activation is dependent on the ambient pressure of the surrounding media, so any increase in hydrostatic pressure demands higher US energies to phase transition. In this paper, the authors explore this basic relationship as a potential direction for noninvasive pressure measurement and foundation of a unique technology the authors are developing termed tumor interstitial pressure estimation using ultrasound (TIPE-US). TIPE-US was developed using a programmable US research scanner. A custom scan sequence interleaved pulsed US transmissions for both PCCA activation and detection. An automated US pressure sweep was applied, and US images were acquired at each increment. Various hydrostatic pressures were applied to PCCA samples. Pressurized samples were imaged using the TIPE-US system. The activation threshold required to convert PCCA from the liquid to gaseous state was recorded for various US and PCCA conditions. Given the relationship between the hydrostatic pressure applied to the PCCA and US energy needed for activation, phase transition can be used as a surrogate of hydrostatic pressure. Consistent with theoretical predictions, the PCCA activation threshold was lowered with increasing sample temperature and by decreasing the frequency of US exposure, but it was not impacted by PCCA concentration.

Ultrasound-Stimulated Drug Delivery Using Therapeutic Reconstituted High-Density Lipoprotein Nanoparticles.

The abnormal tumor vasculature and the resulting abnormal microenvironment are major barriers to optimal chemotherapeutic drug delivery. It is well known that ultrasound (US) can increase the permeability of the tumor vessel walls and enhance the accumulation of anticancer agents. Reconstituted high-density lipoproteins (rHDL) nanoparticles (NPs) allow selective delivery of anticancer agents to tumor cells via their overexpressed scavenger receptor type B1 (SR-B1) receptor. The goal of this study is to investigate the potential of noninvasive US therapy to further improve delivery and tumor uptake of the payload from rHDL NPs, preloaded with an infrared dye (IR-780), aimed to establish a surrogate chemotherapeutic model with optical localization. Athymic nude mice were implanted orthotopically with one million breast cancer cells (MDA-MB-231/Luc). Three weeks later, animals were divided into seven groups with comparable mean tumor size: control, low, moderate, and high concentration of rHDL NPs alone groups, as well as these three levels of rHDL NPs plus US therapy groups (N = 7 to 12 animals per group), where low, moderate and high denote 5, 10, and 50 µg of the IR-780 dye payload per rHDL NP injection, respectively. The US therapy system included a single element focused transducer connected in series with a function generator and power amplifier. A custom 3D printed cone with an acoustically transparent aperture and filled with degassed water allowed delivery of focused US energy to the tumor tissue. US exposure involved a pulsed sequence applied for a duration of 5 min. Each animal in the US therapy groups received a slow bolus co-injection of MB contrast agent and rHDL NPs. Animals were imaged using a whole-body optical system to quantify intratumoral rHDL NP accumulation at baseline and again at 1 min, 30 min, 24 h, and 48 h. At 48 h, all animals were euthanized and tumors were excised for ex vivo analysis. We investigated a noninvasive optical imaging method for monitoring the effects of US-stimulated drug delivery of IR-780 dye-loaded rHDL NPs in living animals. No change in optical imaging data was found in the control animals. However, there was considerable dye accumulation (surrogate drug) within 48 h in the low (5 µg), moderate (10 µg), and high (50 µg) rHDL NP concentration-dosed group animals (p < 0.09). With US therapy added to the experimental protocol, there was an additional and significant increase in local tumor drug uptake at 48 h (p < 0.02). Optical image data collected from ex vivo tumor samples confirmed tumor retention of the IR-780 dye-loaded rHDL NPs and correlated positively with in vivo optical imaging results (R2 > 0.69, p < 0.003). IR-780 dye extraction from the tumor tissue samples confirmed the in vivo and ex vivo US therapy findings. Overall, the addition of US therapy considerably improved local rHDL NP accumulation in tumor tissue. This study concludes that US-mediated drug delivery can facilitate tumor uptake of rHDL NPs and more research is warranted to optimize the drug dosing schedule and the respective therapeutic protocols.

In vivo demonstration of cancer molecular imaging with ultrasound radiation force and buried-ligand microbubbles.

In designing targeted contrast agent materials for imaging, the need to present a targeting ligand for recognition and binding by the target is counterbalanced by the need to minimize interactions with plasma components and to avoid recognition by the immune system. We have previously reported on a microbubble imaging probe for ultrasound molecular imaging that uses a buried-ligand surface architecture to minimize unwanted interactions and immunogenicity. Here we examine for the first time the utility of this approach for in vivo molecular imaging. In accordance with previous results, we showed a threefold increase in circulation persistence through the tumor of a fibrosarcoma model in comparison with controls. The buried-ligand microbubbles were then activated for targeted adhesion through the application of noninvasive ultrasound radiation forces applied specifically to the tumor region. Using a clinical ultrasound scanner, microbubbles were activated, imaged, and silenced. The results showed visually conspicuous images of tumor neovasculature and a twofold increase in ultrasound radiation force enhancement of acoustic contrast intensity for buried-ligand microbubbles, whereas no such increase was found for exposed-ligand microbubbles. We therefore conclude that the use of acoustically active buried-ligand microbubbles for ultrasound molecular imaging bridges the demand for low immunogenicity with the necessity of maintaining targeting efficacy and imaging conspicuity in vivo.

Contrast ultrasound imaging for identification of early responder tumor models to anti-angiogenic therapy.

Agents targeting vascular endothelial growth factor (VEGF) have been validated as cancer therapeutics, yet efficacy can differ widely between tumor types and individual patients. In addition, such agents are costly and can have significant toxicities. Rapid noninvasive determination of response could provide significant benefits. We tested if response to the anti-VEGF antibody bevacizumab (BV) could be detected using contrast-enhanced ultrasound imaging (CEUS). We used two xenograft model systems with previously well-characterized responses to VEGF inhibition, a responder (SK-NEP-1) and a non-responder (NGP), and examined perfusion-related parameters. CEUS demonstrated that BV treatment arrested the increase in blood volume in the SK-NEP-1 tumor group only. Molecular imaging of α(V)ß(3) with targeted microbubbles was a more sensitive prognostic indicator of BV efficacy. CEUS using RGD-labeled microbubbles showed a robust decrease in α(V)ß(3) vasculature following BV treatment in SK-NEP-1 tumors. Paralleling these findings, lectin perfusion assays detected a disproportionate pruning of smaller, branch vessels. Therefore, we conclude that the response to BV can be identified soon after initiation of treatment, often within 3 days, by use of CEUS molecular imaging techniques. The use of a noninvasive ultrasound approach may allow for earlier and more effective determination of efficacy of antiangiogenic therapy.

Monitoring early tumor response to drug therapy with diffuse optical tomography.

Although anti-angiogenic agents have shown promise as cancer therapeutics, their efficacy varies between tumor types and individual patients. Providing patient-specific metrics through rapid noninvasive imaging can help tailor drug treatment by optimizing dosages, timing of drug cycles, and duration of therapy-thereby reducing toxicity and cost and improving patient outcome. Diffuse optical tomography (DOT) is a noninvasive three-dimensional imaging modality that has been shown to capture physiologic changes in tumors through visualization of oxygenated, deoxygenated, and total hemoglobin concentrations, using non-ionizing radiation with near-infrared light. We employed a small animal model to ascertain if tumor response to bevacizumab (BV), an anti-angiogenic agent that targets vascular endothelial growth factor (VEGF), could be detected at early time points using DOT. We detected a significant decrease in total hemoglobin levels as soon as one day after BV treatment in responder xenograft tumors (SK-NEP-1), but not in SK-NEP-1 control tumors or in non-responder control or BV-treated NGP tumors. These results are confirmed by magnetic resonance imaging T2 relaxometry and lectin perfusion studies. Noninvasive DOT imaging may allow for earlier and more effective control of anti-angiogenic therapy.

Theranostic Gd(III)-lipid microbubbles for MRI-guided focused ultrasound surgery.

We have synthesized a biomaterial consisting of Gd(III) ions chelated to lipid-coated, size-selected microbubbles for utility in both magnetic resonance and ultrasound imaging. The macrocyclic ligand DOTA-NHS was bound to PE headgroups on the lipid shell of pre-synthesized microbubbles. Gd(III) was then chelated to DOTA on the microbubble shell. The reaction temperature was optimized to increase the rate of Gd(III) chelation while maintaining microbubble stability. ICP-OES analysis of the microbubbles determined a surface density of 7.5 × 10(5) ± 3.0 × 10(5) Gd(III)/µm(2) after chelation at 50 °C. The Gd(III)-bound microbubbles were found to be echogenic in vivo during high-frequency ultrasound imaging of the mouse kidney. The Gd(III)-bound microbubbles also were characterized by magnetic resonance imaging (MRI) at 9.4 T by a spin-echo technique and, surprisingly, both the longitudinal and transverse proton relaxation rates were found to be roughly equal to that of no-Gd(III) control microbubbles and saline. However, the relaxation rates increased significantly, and in a dose-dependent manner, after sonication was used to fragment the Gd(III)-bound microbubbles into non-gas-containing lipid bilayer remnants. The longitudinal (r(1)) and transverse (r(2)) molar relaxivities were 4.0 ± 0.4 and 120 ± 18 mM(-1)s(-1), respectively, based on Gd(III) content. The Gd(III)-bound microbubbles may find application in the measurement of cavitation events during MRI-guided focused ultrasound therapy and to track the biodistribution of shell remnants.

Advances in ultrasound mediated gene therapy using microbubble contrast agents.

Microbubble ultrasound contrast agents have the potential to dramatically improve gene therapy treatments by enhancing the delivery of therapeutic DNA to malignant tissue. The physical response of microbubbles in an ultrasound field can mechanically perturb blood vessel walls and cell membranes, enhancing drug permeability into malignant tissue. In this review, we discuss literature that provided evidence of specific mechanisms that enhance in vivo gene delivery utilizing microbubble contrast agents, namely their ability to 1) improving cell membrane permeability, 2) modulate vascular permeability, and 3) enhance endocytotic uptake in cells. Additionally, we review novel microbubble vectors that are being developed in order to exploit these mechanisms and deliver higher gene payloads with greater target specificity. Finally, we discuss some future considerations that should be addressed in the development of next-generation microbubbles in order to improve in vivo microbubble gene delivery. Overall, microbubbles are rapidly gaining popularity as efficient gene carriers, and combined with their functionality as imaging contrast agents, they represent powerful theranostic tools for image guided gene therapy applications.

Polyplex-microbubble hybrids for ultrasound-guided plasmid DNA delivery to solid tumors.

Microbubble ultrasound contrast agents are being developed as image-guided gene carriers for targeted delivery in vivo. In this study, novel polyplex-microbubbles were synthesized, characterized and evaluated for systemic circulation and tumor transfection. Branched polyethylenimine (PEI; 25 kDa) was modified with polyethylene glycol (PEG; 5 kDa), thiolated and covalently attached to maleimide groups on lipid-coated microbubbles. The PEI-microbubbles demonstrated increasingly positive surface charge and DNA loading capacity with increasing maleimide content. The in vivo ultrasound contrast persistence of PEI-microbubbles was measured in the healthy mouse kidney, and a two-compartment pharmacokinetic model accounting for free and adherent microbubbles was developed to describe the anomalous time-intensity curves. The model suggested that PEI loading dramatically reduced free circulation and increased nonspecific adhesion to the vasculature. However, DNA loading to form polyplex-microbubbles increased circulation in the bloodstream and decreased nonspecific adhesion. PEI-microbubbles coupled to a luciferase bioluminescence reporter plasmid DNA were shown to transfect tumors implanted in the mouse kidney. Site-specific delivery was achieved using ultrasound applied over the tumor area following bolus injection of the DNA/PEI-microbubbles. In vivo imaging showed over 10-fold higher bioluminescence from the tumor region compared to untreated tissue. Ex vivo analysis of excised tumors showed greater than 40-fold higher expression in tumor tissue than non-sonicated control (heart) tissue. These results suggest that the polyplex-microbubble platform offers improved control of DNA loading and packaging suitable for ultrasound-guided tissue transfection.

Formulation of polylactide-co-glycolic acid nanospheres for encapsulation and sustained release of poly(ethylene imine)-poly(ethylene glycol) copolymers complexed to oligonucleotides.

Antisense oligonucleotides (AOs) have been shown to induce dystrophin expression in muscles cells of patients with Duchenne Muscular Dystrophy (DMD) and in the mdx mouse, the murine model of DMD. However, ineffective delivery of AOs limits their therapeutic potential. Copolymers of cationic poly(ethylene imine) (PEI) and non-ionic poly(ethylene glycol) (PEG) form stable nanoparticles when complexed with AOs, but the positive surface charge on the resultant PEG-PEI-AO nanoparticles limits their biodistribution. We adapted a modified double emulsion procedure for encapsulating PEG-PEI-AO polyplexes into degradable polylactide-co-glycolic acid (PLGA) nanospheres. Formulation parameters were varied including PLGA molecular weight, ester end-capping, and sonication energy/volume. Our results showed successful encapsulation of PEG-PEI-AO within PLGA nanospheres with average diameters ranging from 215 to 240 nm. Encapsulation efficiency ranged from 60 to 100%, and zeta potential measurements confirmed shielding of the PEG-PEI-AO cationic charge. Kinetic measurements of 17 kDa PLGA showed a rapid burst release of about 20% of the PEG-PEI-AO, followed by sustained release of up to 65% over three weeks. To evaluate functionality, PEG-PEI-AO polyplexes were loaded into PLGA nanospheres using an AO that is known to induce dystrophin expression in dystrophic mdx mice. Intramuscular injections of this compound into mdx mice resulted in over 300 dystrophin-positive muscle fibers distributed throughout the muscle cross-sections, approximately 3.4 times greater than for injections of AO alone. We conclude that PLGA nanospheres are effective compounds for the sustained release of PEG-PEI-AO polyplexes in skeletal muscle and concomitant expression of dystrophin, and may have translational potential in treating DMD.