Christopher V. Barback

Therapeutic Enzyme‐Responsive Nanoparticles for Targeted Delivery and Accumulation in Tumors

An enzyme-responsive, paclitaxel-loaded nanoparticle is described and assessed in vivo in a human fibrosarcoma murine xenograft. This work represents a proof-of-concept study demonstrating the utility of enzyme-responsive nanoscale drug carriers capable of targeted accumulation and retention in tumor tissue in response to overexpressed endogenous enzymes.

Enzyme-Directed Assembly of Nanoparticles in Tumors Monitored by in Vivo Whole Animal Imaging and ex Vivo Super-Resolution Fluorescence Imaging

Matrix metalloproteinase enzymes, overexpressed in HT-1080 human fibrocarcinoma tumors, were used to guide the accumulation and retention of an enzyme-responsive nanoparticle in a xenograft mouse model. The nanoparticles were prepared as micelles from amphiphilic block copolymers bearing a simple hydrophobic block and a hydrophilic peptide brush. The polymers were end-labeled with Alexa Fluor 647 dyes leading to the formation of labeled micelles upon dialysis of the polymers from DMSO/DMF to aqueous buffer. This dye-labeling strategy allowed the presence of the retained material to be visualized via whole animal imaging in vivo and in ex vivo organ analysis following intratumoral injection into HT-1080 xenograft tumors. We propose that the material is retained by virtue of an enzyme-induced accumulation process whereby particles change morphology from 20 nm spherical micelles to micrometer-scale aggregates, kinetically trapping them within the tumor. This hypothesis is tested here via an unprecedented super-resolution fluorescence analysis of ex vivo tissue slices confirming a particle size increase occurs concomitantly with extended retention of responsive particles compared to unresponsive controls.

Hollow silica and silica-boron nano/microparticles for contrast-enhanced ultrasound to detect small tumors

Diagnosing tumors at an early stage when they are easily curable and may not require systemic chemotherapy remains a challenge to clinicians. In order to improve early cancer detection, gas filled hollow boron-doped silica particles have been developed, which can be used for ultrasound-guided breast conservation therapy. The particles are synthesized using a polystyrene template and subsequently calcinated to create hollow, rigid nanoporous microspheres. The microshells are filled with perfluoropentane vapor. Studies were performed in phantoms to optimize particle concentration, injection dose, and the ultrasound settings such as pulse frequency and mechanical index. In vitro studies have shown that these particles can be continuously imaged by US up to 48 min and their signal lifetime persisted for 5 days. These particles could potentially be given by intravenous injection and, in conjunction with contrast-enhanced ultrasound, be utilized as a screening tool to detect smaller breast cancers before they are detectible by traditional mammography.

Enzyme‐Directed Assembly of a Nanoparticle Probe in Tumor Tissue

Enzyme-directed assembly in vivo: A targeting strategy is demonstrated, which leads to an active accumulation of nanoparticles by virtue of an assembly event specific to endogenous, enzymatic biochemical signals associated with tumor tissue. The viability of this approach is examined through a proof-of-concept study showing enzyme-directed particle targeting and accumulation in human xenograft tumors in mice following intravenous injection, and the retention of particles is demonstrated within tumors for extended periods of time.

Color Doppler Ultrasound and Gamma Imaging of Intratumorally Injected 500 nm Iron–Silica Nanoshells

Perfluoropentane gas filled iron-silica nanoshells have been developed as stationary ultrasound contrast agents for marking tumors to guide surgical resection. It is critical to establish their long-term imaging efficacy, as well as biodistribution. This work shows that 500 nm Fe-SiO2 nanoshells can be imaged by color Doppler ultrasound over the course of 10 days in Py8119 tumor bearing mice. The 500 nm nonbiodegradable SiO2 and biodegradable Fe-SiO2 nanoshells were functionalized with diethylenetriamine pentaacetic acid (DTPA) ligand and radiolabeled with (111)In(3+) for biodistribution studies in nu/nu mice. The majority of radioactivity was detected in the liver and kidneys following intravenous (IV) administration of nanoshells to healthy animals. By contrast, after nanoshells were injected intratumorally, most of the radioactivity remained at the injection site; however, some nanoshells escaped into circulation and were distributed similarly as those given intravenously. For intratumoral delivery of nanoshells and IV delivery to healthy animals, little difference was seen between the biodistribution of SiO2 and biodegradable Fe-SiO2 nanoshells. However, when nanoshells were administered IV to tumor bearing mice, a significant increase was observed in liver accumulation of SiO2 nanoshells relative to biodegradable Fe-SiO2 nanoshells. Both SiO2 and Fe-SiO2 nanoshells accumulate passively in proportion to tumor mass, during intravenous delivery of nanoshells. This is the first report of the biodistribution following intratumoral injection of any biodegradable silica particle, as well as the first report demonstrating the utility of DTPA-(111)In labeling for studying silica nanoparticle biodistributions.

Neural progenitor cells labeling with microbubble contrast agent for ultrasound imaging in vivo

Tracking neuroprogenitor cells (NPCs) that are used to target tumors, infarction or inflammation, is paramount for cell-based therapy. We employed ultrasound imaging that can detect a single microbubble because it can distinguish its unique signal from those of surrounding tissues. NPCs efficiently internalized positively charged microbubbles allowing a clinical ultrasound system to detect a single cell at 7 MHz. When injected intravenously, labeled NPCs traversed the lungs to be imaged in the left ventricle and the liver where they accumulated. Internalized microbubbles were not only less sensitive to destruction by ultrasound, but remained visible in vivo for days as compared to minutes when given free. The extended longevity provides ample time to allow cells to reach their intended target. We were also able to transfect NPCs in vitro when microbubbles were preloaded with GFP plasmid only when cells were insonated. Transfection efficiency and cell viability were both greater than 90%.

Automating tumor classification with pixel-by-pixel contrast-enhanced ultrasound perfusion kinetics

Contrast-enhanced ultrasound (CEUS) enables highly specific time-resolved imaging of vasculature by intravenous injection of ∼2 μm gas filled microbubbles. To develop a quantitative automated diagnosis of breast tumors with CEUS, breast tumors were induced in rats by administration of N-ethyl-N-nitrosourea. A bolus injection of microbubbles was administered and CEUS videos of each tumor were acquired for at least 3 min. The time-intensity curve of each pixel within a region of interest (ROI) was analyzed to measure kinetic parameters associated with the wash-in, peak enhancement, and wash-out phases of microbubble bolus injections since it was expected that the aberrant vascularity of malignant tumors will result in faster and more diverse perfusion kinetics versus those of benign lesions. Parameters were classified using linear discriminant analysis to differentiate between benign and malignant tumors and improve diagnostic accuracy. Preliminary results with a small dataset (10 tumors, 19 videos) show 100% accuracy with fivefold cross-validation testing using as few as two choice variables for training and validation. Several of the parameters which provided the best differentiation between malignant and benign tumors employed comparative analysis of all the pixels in the ROI including enhancement coverage, fractional enhancement coverage times, and the standard deviation of the envelope curve difference normalized to the mean of the peak frame. Analysis of combinations of five variables demonstrated that pixel-by-pixel analysis produced the most robust information for tumor diagnostics and achieved 5 times greater separation of benign and malignant cases than ROI-based analysis.

In vivo ultrasound visualization of non-occlusive blood clots with thrombin-sensitive contrast agents.

The use of microbubbles as ultrasound contrast agents is one of the primary methods to diagnose deep venous thrombosis. However, current microbubble imaging strategies require either a clot sufficiently large to produce a circulation filling defect or a clot with sufficient vascularization to allow for targeted accumulation of contrast agents. Previously, we reported the design of a microbubble formulation that modulated its ability to generate ultrasound contrast from interaction with thrombin through incorporation of aptamer-containing DNA crosslinks in the encapsulating shell, enabling the measurement of a local chemical environment by changes in acoustic activity. However, this contrast agent lacked sufficient stability and lifetime in blood to be used as a diagnostic tool. Here we describe a PEG-stabilized, thrombin-activated microbubble (PSTA-MB) with sufficient stability to be used in vivo in circulation with no change in biomarker sensitivity. In the presence of actively clotting blood, PSTA-MBs showed a 5-fold increase in acoustic activity. Specificity for the presence of thrombin and stability under constant shear flow were demonstrated in a home-built in vitro model. Finally, PSTA-MBs were able to detect the presence of an active clot within the vena cava of a rabbit sufficiently small as to not be visible by current non-specific contrast agents. By activating in non-occlusive environments, these contrast agents will be able to detect clots not diagnosable by current contrast agents.

Integrated processing of contrast pulse sequencing ultrasound imaging for enhanced active contrast of hollow gas filled silica nanoshells and microshells

In recent years, there have been increasing developments in the field of contrast-enhanced ultrasound both in the creation of new contrast agents and in imaging modalities. These contrast agents have been employed to study tumor vasculature in order to improve cancer detection and diagnosis. An in vivo study is presented of ultrasound imaging of gas filled hollow silica microshells and nanoshells which have been delivered intraperitoneally to an IGROV-1 tumor bearing mouse. In contrast to microbubbles, this formulation of microshells provided strong ultrasound imaging signals by shell disruption and release of gas. Imaging of the microshells in an animal model was facilitated by novel image processing. Although the particle signal could be identified by eye under live imaging, high background obfuscated the particle signal in still images and near the borders of the tumor with live images. Image processing techniques were developed that employed the transient nature of the particle signal to selectively filter out the background signal. By applying image registration, high-pass, median, threshold, and motion filtering, a short video clip of the particle signal was compressed into a single image, thereby resolving the silica shells within the tumor.

Fluorescence-Based Molecular Imaging of Porcine Urinary Bladder Sentinel Lymph Nodes

The primary objective was to test the ability of a laparoscopic camera system to detect the fluorescent signal emanating from sentinel lymph nodes (SLNs) approximately 2 d after injection and imaging of a positron-emitting molecular imaging agent into the submucosa of the porcine urinary bladder. Methods: Three female pigs underwent a submucosal injection of the bladder with fluorescent-tagged tilmanocept, radiolabeled with both 68Ga and 99mTc. One hour after injection, a pelvic PET/CT scan was acquired for preoperative SLN mapping. Approximately 36 h later, robotic SLN mapping was performed using a fluorescence-capable camera system. After identification of the fluorescent lymph nodes, a pelvic lymph node dissection was completed with robotic assistance. All excised nodal packets (n = 36) were assayed for 99mTc activity, which established a lymph node as an SLN. 99mTc activity was also used to calculate the amount of dye within each lymph node. Results: All of the SLNs defined by the ex vivo γ-well assay of 99mTc activity were detected by fluorescence mode imaging. The time between injection and robotic SLN mapping ranged from 32 to 38 h. A total of 5 fluorescent lymph nodes were detected; 2 pigs had 2 fluorescent lymph nodes and 1 pig exhibited a single lymph node. Four of the 5 SLNs exhibited increased SUVs of 12.4–139.0 obtained from PET/CT. The dye content of the injection sites ranged from 371 to 1,441 pmol, which represented 16.5%–64.1% of the injected dose; the amount of dye within the SLNs ranged from 8.5 to 88 pmol, which was equivalent to 0.38%–3.91% of the administered dose. Conclusion: Fluorescent-labeled 68Ga-tilmanocept allows for PET imaging and real-time intraoperative detection of SLNs during robotic surgery.