Michaelann Tartis

Dynamic microPET imaging of ultrasound contrast agents and lipid delivery

Interest in ultrasound contrast agents (lipid-shelled microbubbles) as delivery vehicles is increasing; however, the biodistribution of these agents remains uncharacterized, both with and without ultrasound. In this study, an (18)F-labeled lipid ([(18)F]fluorodipalmitin), incorporated in microbubble shells, was used as a dynamic microPET probe for quantitative 90-minute biodistribution measurements in male Fischer 344 rats (n=2). The spleen retained the highest concentration of radioactive lipid at approximately 2.6%-injected dose per cubic centimeter (% ID/cc) and the liver demonstrated the largest total accumulation (approximately 17% ID). The microbubble pharmacokinetic profile differed from free lipid, which is rapidly cleared from blood, and liposomes, which remain in circulation. Additionally, region of interest (ROI) analysis over 60 minutes (post-ultrasound treatment) quantified the delivery of lipid by therapeutic ultrasound from microbubbles to kidney tissue (n=8). The ultrasound sequence consisted of a 200 kPa, 5.3 MHz radiation force pulse followed by a 1.6 MPa, 1.4 MHz fragmentation pulse and was applied to one kidney, while the contralateral kidney served as a control. ROI-estimated activity in treated kidneys was slightly but significantly greater at 0 and 60 min than in untreated kidneys (p=0.0012 and 0.0035, respectively). This effect increased with the number of microbubbles injected (p=0.006). In summary, [(18)F]fluorodipalmitin was used to characterize the biodistribution of contrast microbubble shells and the deposition of lipid was shown to be locally increased after insonation.

Lipophilic prodrug conjugates allow facile and rapid synthesis of high-loading capacity liposomes without the need for post-assembly purification.

Abstract Dihydropyridopyrazoles are simplified synthetic analogues of podophyllotoxin that can effectively mimic its molecular scaffold and act as potent mitotic spindle poisons in dividing cancer cells. However, despite nanomolar potencies and ease of synthetic preparation, further clinical development of these promising anticancer agents is hampered due to their poor aqueous solubility. In this article, we developed a prodrug strategy that enables incorporation of dihydropyridopyrazoles into liposome bilayers to overcome the solubility issues. The active drug was covalently connected to either myristic or palmitic acid anchor via carboxylesterase hydrolyzable linkage. The resulting prodrugs were self-assembled into liposome bilayers from hydrated lipid films using ultrasound without the need for post-assembly purification. The average particle size of the prodrug-loaded liposomes was about 90 nm. The prodrug incorporation was verified by differential scanning calorimetry, spectrophotometry and gel filtration reaching maximum at 0.3 and 0.35 prodrug/lipid molar ratios for myristic and palmitic conjugates, respectively. However, the ratio of 0.2 was used in the particle size and biological activity experiments to maintain long-term stability of the prodrug-loaded liposomes against phase separation during storage. Antiproliferative activity was tested against HeLa and Jurkat cancer cell lines in vitro showing that the liposomal prodrug retained antitubulin activity of the parent drug and induced apoptosis-mediated cancer cell death. Overall, the established data provide a powerful platform for further clinical development of dihydropyridopyrazoles using liposomes as the drug delivery system.

Experimental Study of the Mechanics of Blast-Induced Traumatic Brain Injury

Blast-induced traumatic brain injury (bTBI) has become a “signature wound” of modern combat due to the expansive use of improvised explosive devices (IED). bTBI is characterized as either primary, secondary, tertiary, or quanternary.

Control over Silica Particle Growth and Particle-Biomolecule Interactions Facilitates Silica Encapsulation of Mammalian Cells with Thickness Control

Over the last twenty years, many strategies utilizing sol-gel chemistry to integrate biological cells into silica-based materials have been reported. One such strategy, Sol-Generating Chemical Vapor into Liquid (SG-CViL) deposition, shows promise as an efficient encapsulation technique due to the ability to vary the silica encapsulation morphology obtained by this process through variation of SG-CViL reaction conditions. In this report, we develop SG-CViL as a tunable, multi-purpose silica encapsulation strategy by investigating the mechanisms governing both silica particle generation and subsequent interaction with phospholipid assemblies (liposomes and living cells). Using Dynamic Light Scattering (DLS) measurements, linear and exponential silica particle growth dynamics were observed which were dependent on deposition buffer ion constituents and ion concentration. Silica particle growth followed a cluster-cluster growth mechanism at acidic pH, and a monomer-cluster growth mechanism at neutral to basic pH. Increasing silica sol aging temperature resulted in higher rates of particle growth and larger particles. DLS measurements employing PEG coated liposomes and cationic liposomes, serving as model phospholipid assemblies, revealed electrostatic interactions promote more stable liposome-silica interactions than hydrogen bonding and facilitate silica coating on suspension cells. However, continued silica reactivity leads to aggregation of silica coated suspensions cells, revealing the need for cell isolation to tune deposited silica thickness. Utilizing these mechanistic study insights, silica was deposited onto adherent HeLa cells under biocompatible conditions with micron scale control over silica thickness, minimal cell manipulation steps, and retained cell viability over several days.

Sol-Generating Chemical Vapor into Liquid (SG-CViL) deposition – a facile method for encapsulation of diverse cell types in silica matrices

In nature, cells perform a variety of complex functions such as sensing, catalysis, and energy conversion which hold great potential for biotechnological device construction. However, cellular sensitivity to ex-vivo environments necessitates development of bio-nano interfaces which allow integration of cells into devices and maintain their desired functionality. In order to develop such an interface, the use of a novel Sol Generating Chemical Vapor into Liquid (SG-CViL) deposition process for whole cell encapsulation in silica was explored. In SG-CViL, the high vapor pressure of tetramethyl orthosilicate (TMOS) is utilized to deliver silica into an aqueous medium, creating a silica sol. Cells are then mixed with the resulting silica sol, facilitating encapsulation of cells in silica while minimizing cell contact with the cytotoxic products of silica generating reactions (i.e. methanol), and reduce exposure of cells to compressive stresses induced from silica condensation reactions. Using SG-CVIL, Saccharomyces cerevisiae (S. cerevisiae) engineered with an inducible beta galactosidase system were encapsulated in silica solids and remained both viable and responsive 29 days post encapsulation. By tuning SG-CViL parameters thin layer silica deposition on mammalian HeLa and U87 human cancer cells was also achieved. The ability to encapsulate various cell types in either a multi cell (S. cerevisiae) or a thin layer (HeLa and U87 cells) fashion shows the promise of SG-CViL as an encapsulation strategy for generating cell-silica constructs with diverse functions for incorporation into devices for sensing, bioelectronics, biocatalysis, and biofuel applications.

Pharmacokinetics of Encapsulated Paclitaxel: Multi-Probe Analysis With PET

We have combined two imaging probes and used PET as a means to provide image-based validation for a novel targeted drug delivery system. The first probe was a direct labeling of the drug [18F]fluoropaclitaxel [1–3], which was inserted into various carrier vehicle formulations. The second probe, [18F]fluoro-1,2-dipalmitoyl-sn-glycerol, i.e. [18F]FDP involved radiolabeling the lipid vehicle. Paclitaxel, which is poorly soluble in aqueous media, also has limited solubility and stability in lipophilic environments such as liposomes. Stable association of paclitaxel with the lipid bilayer is affected by a variety of physicochemical factors such as temperature and liposome composition. Paclitaxel crystal formation has been documented, with two forms of solid state within aqueous media and organic solvents, although crystal conformation differs in each media [4,5]. We provide dynamic in vivo image sets providing biodistribution and time activity curves of free [18F]fluoropaclitaxel and liposomal [18F]fluoropaclitaxel as well as free [18F]FDP, liposomal [18F]FDP, and [18F]FDP in an ultrasound contrast agent. Serial studies were performed within a small group of rats, minimizing inter-animal variability. The two labeled molecules have different biodistributions: paclitaxel is rapidly taken up in the liver, intestines and kidneys, while the labeled lipid incorporated into liposomes stays in circulation with minimal uptake in organs other than spleen. Here, we have developed a quantitative method to follow paclitaxel and lipid vehicles to their destination in vivo in order to improve targeted paclitaxel delivery.Copyright

Enhanced drug delivery with ultrasound and engineered delivery vehicles

We demonstrate that local drug delivery can be achieved by ultrasound, combined with engineered delivery vehicles, where the vehicles have a diameter on the order of nanometers to microns. Delivery vehicles can be created from microbubbles with a thickened shell or a lipid shell decorated with drugs, genes, or nanoparticles. Alternatively, liquid‐filled nanoparticles can be employed to carry the desired compound. Ultrasound can deflect these vehicles from the center of the flowstream, can fragment the vehicle releasing its contents, and may enhance the uptake of the particle or its contents by cells in the desired region. The ultrasonic mechanisms behind these changes are summarized. The addition of targeting ligands to the shell to improve target specificity is also explored. Methods to measure the effectiveness of local drug delivery, including correlative imaging modalities, binding assays, and cytotoxicity assays, will be described. [The support of NIH CA 103828 is gratefully acknowledged.]