Praveena Mohan

Ultrasound-Mediated Tumor Imaging and Nanotherapy using Drug Loaded, Block Copolymer Stabilized Perfluorocarbon Nanoemulsions

Perfluorocarbon nanoemulsions can deliver lipophilic therapeutic agents to solid tumors and simultaneously provide for monitoring nanocarrier biodistribution via ultrasonography and/or (19)F MRI. In the first generation of block copolymer stabilized perfluorocarbon nanoemulsions, perfluoropentane (PFP) was used as the droplet forming compound. Although manifesting excellent therapeutic and ultrasound imaging properties, PFP nanoemulsions were unstable at storage, difficult to handle, and underwent hard to control phenomenon of irreversible droplet-to-bubble transition upon injection. To solve the above problems, perfluoro-15-crown-5-ether (PFCE) was used as a core forming compound in the second generation of block copolymer stabilized perfluorocarbon nanoemulsions. PFCE nanodroplets manifest both ultrasound and fluorine ((19)F) MR contrast properties, which allows using multimodal imaging and (19)F MR spectroscopy for monitoring nanodroplet pharmacokinetics and biodistribution. In the present paper, acoustic, imaging, and therapeutic properties of unloaded and paclitaxel (PTX) loaded PFCE nanoemulsions are reported. As manifested by the (19)F MR spectroscopy, PFCE nanodroplets are long circulating, with about 50% of the injected dose remaining in circulation 2h after the systemic injection. Sonication with 1-MHz therapeutic ultrasound triggered reversible droplet-to-bubble transition in PFCE nanoemulsions. Microbubbles formed by acoustic vaporization of nanodroplets underwent stable cavitation. The nanodroplet size (200nm to 350nm depending on a type of the shell and conditions of emulsification) as well as long residence in circulation favored their passive accumulation in tumor tissue that was confirmed by ultrasonography. In the breast and pancreatic cancer animal models, ultrasound-mediated therapy with paclitaxel-loaded PFCE nanoemulsions showed excellent therapeutic properties characterized by tumor regression and suppression of metastasis. Anticipated mechanisms of the observed effects are discussed.

Doxorubicin as a molecular nanotheranostic agent: effect of doxorubicin encapsulation in micelles or nanoemulsions on the ultrasound-mediated intracellular delivery and nuclear trafficking

Doxorubicin (DOX) is one of the most commonly used chemotherapeutic drugs and is a popular research tool due to the inherent fluorescence of the DOX molecule. After DOX injection, fluorescence imaging of organs or cells can provide information on drug biodistribution. Therapeutic and imaging capabilities combined in a DOX molecule make it an excellent theranostic agent. However, DOX fluorescence depends on a number of factors that should be taken into consideration when interpreting results of DOX fluorescence measurements. Discussing these problems is the main thrust of the current paper. The sensitivity of DOX fluorescence intensity to DOX concentration, local microenvironment, and interaction with model cellular components is illustrated by fluorescence spectra of paired DOX/phospholipid, DOX/histone, DOX/DNA, and triple DOX/histone/DNA and DOX/phospholipid/DNA systems. DOX fluorescence is dramatically quenched upon intercalation into the DNA; DOX fluorescence is also self-quenched at high concentrations of molecularly dissolved DOX; in contrast, DOX fluorescence is increased after binding to the histone or partitioning into the phospholipid phase of PEG-phospholipid micelles or hydrophobic cores of polymeric micelles. While flow cytometry is commonly used for characterization of DOX intracellular uptake, the above aspects of DOX fluorescence may significantly complicate interpretation of flow cytometry results. High cell fluorescence measured by flow cytometry may provide deceptive information on the actual intracellular DOX concentration and may not correlate with the therapeutic efficacy if DOX does not penetrate into the site of action in cell nuclei. These problems are illustrated in the experiments on the intracellular trafficking of DOX encapsulated in poly(ethylene glycol)-co-polycaprolactone (PEG-PCL) micelles or PEG-PCL stabilized perfluorocarbon nanodroplets, with and without the application of ultrasound used as an external trigger. For efficient encapsulation in micelle cores, DOX is usually deprotonated, which removes the positive charge and enhances hydrophobicity of DOX molecule. It was found that the deprotonated DOX accumulated in the cell cytoplasm but did not penetrate into the cell nuclei. The same was true for the DOX encapsulated in micelles or nanodroplets, which may explain their low therapeutic efficacy in the absence of ultrasound. Ultrasound triggers DOX trafficking into the cell nuclei, which is especially pronounced in the presence of nanoemulsions that convert into microbubbles under the ultrasound action. Microbubble cavitation results in the transient permeabilization of both plasma and nuclear membranes, thus allowing DOX penetration into the cell nuclei, which dramatically enhances therapeutic efficacy of DOX-loaded nanodroplet systems.

Comparison of Surfactants Used to Prepare Aqueous Perfluoropentane Emulsions for Pharmaceutical Applications

Perfluoropentane (PFP), a very hydrophobic, nontoxic, noncarcinogenic fluoroalkane, has generated much interest in biomedical applications, including occlusion therapy and controlled drug delivery. For most of these applications, the dispersion within aqueous media of a large quantity of PFP droplets of the proper size is critically important. Surprisingly, the interfacial tension of PFP against water in the presence of surfactants used to stabilize the emulsion has rarely, if ever, been measured. In this study, we report the interfacial tension of PFP in the presence of surfactants used in previous studies to produce emulsions for biomedical applications: polyethylene oxide-co-polylactic acid (PEO-PLA) and polyethylene oxide-co-poly-epsilon-caprolactone (PEO-PCL). Because both of these surfactants are uncharged diblock copolymers that rely on the mechanism of steric stabilization, we also investigate for comparisons sake the use of the small-molecule cationic surfactant cetyl trimethyl ammonium bromide (CTAB) and the much larger protein surfactant bovine serum albumin (BSA). The results presented here complement previous reports of the PFP droplet size distribution and will be useful for determining to what extent the interfacial tension value can be used to control the mean PFP droplet size.

Selective Vaporization of Superheated Nanodroplets for Rapid, Sensitive, Acoustic Biosensing

Superheated perfluorocarbon nano-droplets exhibit promise as sensitive acoustic biosensors. Aggregation of biotin-decorated lipid-shelled droplets by streptavidin greatly increases the yield of bubbles formed by ultrasound-induced vaporization. Streptavidin is sensed down to 1 × 10(-13) m, with differentiable signal appearing in as little as two minutes, using a scalable assay without washing, processing, or development steps.

Direct conjugation of DNA to quantum dots for scalable assembly of photoactive thin films

For many thin film applications, it is critical to not only control the organization of the materials on surfaces but to also use scalable processes that are time and material efficient. This is especially the case when using nanomaterials, including metal or semiconductor nanocrystals, as building blocks from which to engineer devices. In this work, we demonstrate a method to directly conjugate DNA to cadmium based quantum dots (QD) to create thin film arrays on surfaces for potential optoelectronic devices. These DNA-conjugated QDs showed uniform coatings, were oxidation-stable, and remained stable in high ionic strength environments. In previously published work, we discovered that high salt, in particular magnesium, is critical for fabricating nanoparticle assemblies on substrates through DNA interactions. The QD thin films were produced by means of interparticle DNA hybridization in a few steps with no loss of material and with good control over film thickness and roughness. By directly conjugating the DNA to the QDs, it also became possible to study DNAs role in mediating charge transport in the QD films. For this, DNA-conjugated CdTe nanocrystals were assembled onto TiO2 films to fabricate photovoltaic prototypes. Current–Voltage measurements from the DNA–QD devices showed the promise of using DNA not only as an assembler but also as mediator of charge separation and transport.

On-Demand Droplet Fusion: A Strategy for Stimulus-Responsive Biosensing in Solution

A novel strategy is reported for biochemically controlled fusion of oil-in-water (O/W) droplets as an in-solution sensor for biological targets. Inspired by the SNARE complex in cells, the emulsions were stabilized by a combination of phospholipids, phospholipid–poly(ethylene glycol) conjugates, and cholesterol-anchored oligonucleotides. Prior to oligonucleotide binding, the droplets were stable in aqueous media, but hybridization of the oligonucleotides in a zipperlike fashion was shown to initiate droplet fusion. Using image analysis of content mixing of dye-loaded droplets, fusion specificity was studied and optimized as a function of interfacial chemistry. Changing the orientation of the anchored oligonucleotides, using long-chain phospholipids (C18 and C22), and binding a complementary oligonucleotide slowed or even halted fusion completely. Based on these studies, a sensor for the biomarker thrombin was designed using competitive binding of aptamer strands, with droplet fusion increasing as a function of thrombin addition in accordance with a simple binding model, with sensitivity down to 100 nM and with results in as little as 15 min. Future efforts will focus on utilizing this mechanism of content mixing to facilitate highly sensitive detection via modalities such as magnetoresistance or chemiluminescence.

Overcoming Biological Barriers with Ultrasound.

Effect of ultrasound on the permeability of blood vessels and cell membranes to macromolecules and nanodroplets was investigated using mouse carotid arteries and tumor cells. Model macromolecular drug, FITC-dextran with molecular weight of 70,000 Da was used in experiments with carotid arteries. The effect of unfocused 1-MHz ultrasound and and perfluoro-15-crown-5-ether nanodroplets stabilized with the poly(ethylene oxide)-co-poly(D,L-lactide) block copolymer shells was studied. In cell culture experiments, ovarian carcinoma cells and Doxorubicin (DOX) loaded poly(ethylene oxide)-co-polycaprolactone nanodroplets were used. The data showed that the application of ultrasound resulted in permeabilization of all biological barriers tested. Under the action of ultrasound, not only FITC-dextran but also nanodroplets effectively penetrated through the arterial wall; the effect of continuous wave ultrasound was stronger than that of pulsed ultrasound. In cell culture experiments, ultrasound triggered DOX penetration into cell nuclei, presumably due to releasing the drug from the carrier. Detailed mechanisms of the observed effects require further study.