Drug-Loaded Perfluorocarbon Nanodroplets for Ultrasound-Mediated Drug Delivery.

The interaction of nanoparticles with directed energy is a novel application in targeted drug delivery. This chapter focuses on perfluorocarbon nanoemulsions, whose action in drug delivery depends on the ultrasound-triggered phase shift from liquid to gaseous state. These nanoemulsions have great potential for unloading encapsulated drugs at a desired time and location in the body in response to directed ultrasound. In addition, they actively alter their nano-environment for enhancing drug transport through various biological barriers to sites of action, which significantly enhances therapeutic outcome.

Ultrasound-mediated micellar drug delivery.

During the last decade, nanomedicine has emerged as a new field of medicine that utilises nanoscale materials for delivery of drugs, genes and imaging agents. The efficiency of drug delivery may be enhanced by the application of directed energy, which provides for drug targeting and enhanced intracellular uptake. In this paper, we present a review of recent advances in the ultrasound-mediated drug delivery with the emphasis on polymeric micelles as tumour-targeted drug carriers. This new modality of drug targeting to tumours is based on the drug encapsulation in polymeric micelles followed by a localised release at the tumour site triggered by focused ultrasound. The rationale behind this approach is that drug encapsulation in micelles decreases systemic concentration of free drug and provides for a passive drug targeting to tumours via the enhanced permeability and retention (EPR) effect, therefore reducing unwanted drug interactions with healthy tissues. Ultrasound affects micellar drug delivery on various levels. Mild hyperthermia induced by ultrasound may enhance micelle extravasation into tumour tissue; mechanical action of ultrasound results in drug release from micelles and enhances the intracellular uptake of both released and encapsulated drug. In addition, polymeric micelles sensitise multidrug resistant (MDR) cells to the action of drugs.

Genexol inhibits primary tumour growth and metastases in gemcitabine-resistant pancreatic ductal adenocarcinoma.

INTRODUCTION: Gemcitabine, the current standard of care for pancreatic ductal adenocarcinoma (PDA), has a less than 10% partial response rate. Genexol-PM, a modified form of paclitaxel, has been shown to have antitumour effects in clinical trials of metastatic breast and small-lung-cell carcinoma. The aim of the present study was to determine if Genexol would be a beneficial treatment for gemcitabine-resistant PDA. MATERIALS AND METHODS: We measured the in vitro IC50s of gemcitabine and genexol in cell lines sensitive and resistant to gemcitabine. In vivo, animals with orthotopic pancreatic tumours, resistant to gemcitabine, were treated with phosphate-buffered saline (PBS), gemcitabine, Genexol or gemcitabine+Genexol. Tumour progression was monitored using red fluorescent protein imaging. RESULTS: We showed equivalent IC50s for gemcitabine-sensitive and gemcitabine-resistant cell lines when treated with genexol. In vivo treatment with genexol resulted in a greater per cent reduction in tumour size, less metastatic spread and longer survival compared with treatment with gemcitabine. DISCUSSION: Genexol proved to be an effective treatment for gemcitabine-resistant PDA. These data combined with the successful clinical use of genexol in Phase II trials of other malignancies suggests it maybe an effective treatment for pancreatic cancer, specifically for those patients resistant to gemcitabine.

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.

Ultrasonic nanotherapy of pancreatic cancer: lessons from ultrasound imaging.

Pancreatic ductal adenocarcinoma (PDA) is the fourth most common cause of cancer death in the United States, with a median survival time of only 3-6 months for forty percent of patients. Current treatments are ineffective, and new PDA therapies are urgently needed. In this context, ultrasound-mediated chemotherapy by polymeric micelles and/or nanoemulsion/microbubble encapsulated drugs may offer an innovative approach to PDA treatment. PDA xenografts were orthotopically grown in the pancreas tails of nu/nu mice by surgical insertion of red fluorescence protein (RFP)-transfected MiaPaCa-2 cells. Tumor growth was controlled by fluorescence imaging. Occasional sonographic measurements correlated well with the formal tumor tracking by red fluorescence. Tumor accumulation of paclitaxel-loaded nanoemulsion droplets and droplet-to-bubble transition under therapeutic ultrasound was monitored by diagnostic ultrasound imaging. MiaPaCa-2 tumors manifested resistance to treatment by gemcitabine (GEM). This drug is the gold standard for PDA therapy. The GEM-resistant tumors proved sensitive to paclitaxel. Among six experimental groups studied, the strongest therapeutic effect was exerted by the following drug formulation: GEM + nanodroplet-encapsulated paclitaxel (nbGEN) combined with tumor-directed 1-MHz ultrasound that was applied for 30 s four to five hours after the systemic drug injection. Ultrasound-mediated PDA therapy by either micellar or nanoemulsion encapsulated paclitaxel resulted in substantial suppression of metastases and ascites, suggesting ultrasound-enhanced killing of invasive cancerous cells. However, tumors relapsed after the completion of therapy, indicating survival of some tumor cells. The recurrent tumors manifested development of paclitaxel resistance. Ultrasound imaging suggested nonuniform distribution of nanodroplets in the tumor volume due to irregular vascularization, which may result in the development of zones with subtherapeutic drug concentration. This is implicated as a possible cause of the resistance development, which may be pertinent to various modes of tumor nanotherapy.

[Recent advances in the applications of ultrasonic microbubbles as gene or drug vectors].

With the research and application of the new ultrasound microbubble contrast agents, ultrasonic microbubbles can not only help to image, but they can also be used as genes or drug carriers. The microbubbles as genes or drug carriers can pass across the endothelial cell barrier and release genes and drug under the action of ultrasound field, which achieve target treatment effect. Based on the relevant materials, the bioeffects, early successes with gene and drug delivery, and potential clinical applications are reviewed.

Controlled and targeted tumor chemotherapy by micellar-encapsulated drug and ultrasound.

The results of a comprehensive in vivo study of a novel tumor-targeting modality are reported. The technique utilized in this study is based on the encapsulation of the chemotherapeutic agent within polymeric micelles in combination with a local ultrasonic irradiation of the tumor. A doxorubicin (DOX) biodistribution, a yield of the internal tumors and a growth rate of the subcutaneous (s.c.) tumors was compared for molecularly dissolved and micellar-encapsulated DOX. This was done with and without tumor sonication, using an ovarian carcinoma tumor model in nu/nu mice. Pure and mixed Pluronic P-105, PEG2000-diacylphospholipid, and poly(ethylene glycol)-co-poly(beta-benzyl-L-aspartate) micelles were used as drug carriers. DOX intracellular uptake was characterized by flow cytometry. A local ultrasonic irradiation of the tumor resulted in a substantially increased drug accumulation in the tumor cells. The effect of the ultrasound was dependent on the time between ultrasound application and drug injection. Ultrasound did not enhance micelle extravasation; the ultrasonic enhancement of drug internalization by the tumor cells required a preliminary passive drug accumulation in the tumor interstitium. Due to the ultrasound-enhanced drug intracellular uptake and cell killing, the yield of intraperitoneal (i.p.) ovarian carcinoma tumors decreased from 70% for DOX dissolved in PBS (positive control) to 36% for the same concentration of DOX encapsulated in Pluronic micelles combined with a 30-s sonication of the abdominal region of a mouse (3 mg/kg DOX, i.p. injection 1 day after inoculation, n>or=10). For s.c. tumors, micellar delivery combined with localized ultrasonic tumor irradiation resulted in a substantial decrease of the tumor growth rates compared to a positive control (3 mg/kg DOX, i.v. injections, n=7, p<0.05). Possible mechanisms of the ultrasound bioeffects on in vivo drug targeting are discussed.

Polymeric micelles and nanoemulsions as tumor-targeted drug carriers: Insight through intravital imaging.

Intravital imaging of nanoparticle extravasation and tumor accumulation has revealed, for the first time, detailed features of carrier and drug behavior in circulation and tissue that suggest new directions for optimization of drug nanocarriers. Using intravital fluorescent microscopy, the extent of the extravasation, diffusion in the tissue, internalization by tissue cells, and uptake by the RES system were studied for polymeric micelles, nanoemulsions, and nanoemulsion-encapsulated drug. Discrimination of vascular and tissue compartments in the processes of micelle and nanodroplet extravasation and tissue accumulation was possible. A simple 1-D continuum model was suggested that allowed discriminating between various kinetic regimes of nanocarrier (or released drug) internalization in tumors of various sizes and cell density. The extravasation and tumor cell internalization occurred much faster for polymeric micelles than for nanoemulsion droplets. Fast micelle internalization resulted in the formation of a perivascular fluorescent coating around blood vessels. A new mechanism of micelle extravasation and internalization was suggested, based on the fast extravasation and internalization rates of copolymer unimers while maintaining micelle/unimer equilibrium in the circulation. The data suggested that to be therapeutically effective, nanoparticles with high internalization rate should manifest fast diffusion in the tumor tissue in order to avoid generation of concentration gradients that induce drug resistance. However an extra-fast diffusion should be avoided as it may result in the flow of extravasated nanoparticles from the tumor to normal organs, which would compromise targeting efficiency. The extravasation kinetics were different for nanodroplets and nanodroplet-encapsulated drug F-PTX suggesting a premature release of some fraction of the drug from the carrier. In conclusion, the development of an "ideal" drug carrier should involve the optimization of both drug retention and carrier diffusion parameters.

Polymeric micelles and nanoemulsions as drug carriers: Therapeutic efficacy, toxicity, and drug resistance.

The manuscript reports the side-by-side comparison of therapeutic properties of polymeric micelles and nanoemulsions generated from micelles. The effect of the structure of a hydrophobic block of block copolymer on the therapeutic efficacy, tumor recurrence, and development of drug resistance was studied in pancreatic tumor bearing mice. Mice were treated with paclitaxel (PTX) loaded poly(ethylene oxide)-co-polylactide micelles or corresponding perfluorocarbon nanoemulsions. Two structures of the polylactide block differing in a physical state of micelle cores or corresponding nanodroplet shells were compared. Poly(ethylene oxide)-co-poly(d,l-lactide) (PEG-PDLA) formed micelles with elastic amorphous cores while poly(ethylene oxide)-co-poly(l-lactide) (PEG-PLLA) formed micelles with solid crystalline cores. Micelles and nanoemulsions stabilized with PEG-PDLA copolymer manifested higher therapeutic efficacy than those formed with PEG-PLLA copolymer studied earlier. Better performance of PEG-PDLA micelles and nanodroplets was attributed to the elastic physical state of micelle cores (or droplet shells) allowing adequate rate of drug release via drug diffusion and/or copolymer biodegradation. The biodegradation of PEG-PDLA stabilized nanoemulsions was monitored by the ultrasonography of nanodroplets injected directly into the tumor; the PEG-PDLA stabilized nanodroplets disappeared from the injection site within 48h. In contrast, nanodroplets stabilized with PEG-PLLA copolymer were preserved at the injection site for weeks and months indicating extremely slow biodegradation of solid PLLA blocks. Multiple injections of PTX-loaded PEG-PDLA micelles or nanoemulsions to pancreatic tumor bearing mice resulted in complete tumor resolution. Two of ten tumors treated with either PEG-PDLA micellar or nanoemulsion formulation recurred after the completion of treatment but proved sensitive to the second treatment cycle indicating that drug resistance has not been developed. This is in contrast to the treatment with PEG-PLLA micelles or nanoemulsions where all resolved tumors quickly recurred after the completion of treatment and proved resistant to the repeated treatment. The prevention of drug resistance in tumors treated with PEG-PDLA stabilized formulations was attributed to the presence and preventive effect of copolymer unimers that were in equilibrium with PEG-PDLA micelles. PEG-PDLA stabilized nanoemulsions manifested lower hematological toxicity than corresponding micelles suggesting higher drug retention in circulation. Summarizing, micelles with elastic cores appear preferable to those with solid cores as drug carriers. Micelles with elastic cores and corresponding nanoemulsions both manifest high therapeutic efficacy, with nanoemulsions exerting lower systemic toxicity than micelles. The presence of a small fraction of micelles with elastic cores in nanoemulsion formulations is desirable for prevention of the development of drug resistance.

Drug delivery in polymeric micelles: from in vitro to in vivo.

A new drug delivery modality was developed based on drug encapsulation in polymeric micelles followed by a controlled release at the tumor site triggered by ultrasound focused on the tumor. Ultrasound not only released drug from micelles but also enhanced the local uptake of both free and encapsulated drug by tumor cells, thus providing effective drug targeting. The significant success of in vitro studies of this new drug delivery technique warranted extending studies to animal experiments. Here the results of the in vitro studies of the above technique are summarized and the first in vivo experiments using colon cancer model in rats are reported. The in vivo results showed that application of low-frequency ultrasound (20 and 70 kHz) significantly reduced the tumor size when compared with non-insonated controls; this result indicated in vivo drug targeting to tumors by ultrasound.

Drug delivery in pluronic micelles: effect of high-frequency ultrasound on drug release from micelles and intracellular uptake.

The effect of high-frequency ultrasound on doxorubicin (DOX) release from Pluronic micelles and intracellular DOX uptake was studied for promyelocytic leukemia HL-60 cells, ovarian carcinoma drug-sensitive and multidrug-resistant (MDR) cells (A2780 and A2780/ADR, respectively), and breast cancer MCF-7 cells. Cavitation events initiated by high-frequency ultrasound were recorded by radical trapping. The onset of transient cavitation and DOX release from micelles were observed at much higher power densities than at low-frequency ultrasound (20-100 kHz). Even a short (15-30 s) exposure to high-frequency ultrasound significantly enhanced the intracellular DOX uptake from PBS, RPMI 1640, and Pluronic micelles. The mechanisms of the observed effects are discussed.

Ultrasonic release of doxorubicin from Pluronic P105 micelles stabilized with an interpenetrating network of N,N-diethylacrylamide.

Pluronic P105 micelles sequester hydrophobic drugs and release them upon insonation with low frequency ultrasound; however these micelles dissolve relatively quickly upon dilution. The objective of this research was to determine whether stabilization of these micelles would compromise their ability to sequester and release drug. P105 micelles were stabilized with an interpenetrating network of poly (N,N-diethylacrylamide), and ultrasonically-activated release of doxorubicin (Dox) was measured by a fluorescence technique. Results showed that stabilized micelles sequestered the Dox and released it upon insonation at 70 kHz. The amount released was not significantly different from that released from P105 micelles (P=0.481), and the drug re-encapsulation upon cessation of insonation was complete. This system has potential for controlled drug delivery to insonated tissues in vivo.

Intracellular uptake and trafficking of Pluronic micelles in drug-sensitive and MDR cells: effect on the intracellular drug localization.

The intracellular uptake and localization of a fluorescently labeled Pluronic P-105 in HL-60 leukemia cells and in A2780 drug-sensitive and A2780/ADR MDR ovarian carcinoma cells were characterized by flow cytometry and fluorescence microscopy. Pluronic P-105 molecules were labeled with a pH-sensitive fluorescent label, 5-(and 6-)carboxy-2'7'-dichlorofluorescein. The fluorescence intensity of labeled Pluronic was about twofold higher at pH 7.4 than at pH 5.5. At Pluronic concentrations exceeding the critical micelle concentration (cmc), flow cytometry histograms manifested bimodal distribution of cell fluorescence for all types of cells. Cell population characterized by higher fluorescence intensity presumably resulted from Pluronic transfer from the acidic environment of cytoplasmic vesicles (endosomes or lysosomes) into the neutral environment of the cytoplasm and cell nuclei, which suggested the permeabilization of the membranes of acidic vesicle by Pluronic molecules. For the MDR cells, the bimodal distribution of cell fluorescence was already observed at very low Pluronic concentrations in the incubation medium (i.e., below the cmc). The data suggest that the membranes of acidic vesicles of MDR cells are more susceptible to the action of polymeric surfactants than those of drug-sensitive cells. Permeabilization of acidic vesicles had a dramatic effect on the intracellular trafficking of drugs: when delivered in PBS, the anthracyclin drug ruboxyl (Rb) sequestered in cytoplasmic vesicles and was excluded from cell nuclei; however, when delivered in Pluronic micelles, drug accumulated in cell nuclei. Drug uptake from/with Pluronic micelles was substantially enhanced by ultrasound. These findings suggest that the nuclear accumulation of drugs internalized via fluid-phase endocytosis can be enhanced by the application of Pluronic micelles and can be further augmented by ultrasonic irradiation.

Effect of ultrasound on the permeability of vascular wall to nano-emulsion droplets.

The effect of ultrasound on the permeability of blood vessels to nano-emulsion droplets was investigated using excised mouse carotid arteries as model blood vessels. Perfluorocarbon nano-droplets were formed by perfluoro-15-crown-5-ether and stabilized by poly(ethylene oxide)-co-poly(DL-lactide) block co-polymer shells. Nano-droplet fluorescence was imparted by interaction with fluorescein isothiocyanate-dextran (molecular weight = 70,000 Da). The permeability of carotid arteries to nano-droplets was studied in the presence and absence of continuous wave or pulsed therapeutic 1-MHz ultrasound. The data indicated that the application of ultrasound resulted in permeabilization of the vascular wall to nano-droplets. The effect of continuous wave ultrasound was substantially stronger than that of pulsed ultrasound of the same total energy. No effect of blood vessel pre-treatment with ultrasound was observed.

Focused ultrasound-mediated drug delivery to pancreatic cancer in a mouse model.

BACKGROUND: Many aspects of the mechanisms involved in ultrasound-mediated therapy remain obscure. In particular, the relative roles of drug and ultrasound, the effect of the time of ultrasound application, and the effect of tissue heating are not yet clear. The current study was undertaken with the goal to clarify these aspects of the ultrasound-mediated drug delivery mechanism. METHODS: Focused ultrasound-mediated drug delivery was performed under magnetic resonance imaging guidance (MRgFUS) in a pancreatic ductal adenocarcinoma (PDA) model grown subcutaneously in nu/nu mice. Paclitaxel (PTX) was used as a chemotherapeutic agent because it manifests high potency in the treatment of gemcitabine-resistant PDA. Poly(ethylene oxide)-co-poly(d,l-lactide) block copolymer stabilized perfluoro-15-crown-5-ether nanoemulsions were used as drug carriers. MRgFUS was applied at sub-ablative pressure levels in both continuous wave and pulsed modes, and only a fraction of the tumor was treated. RESULTS: Positive treatment effects and even complete tumor resolution were achieved by treating the tumor with MRgFUS after injection of nanodroplet encapsulated drug. The MRgFUS treatment enhanced the action of the drug presumably through enhanced tumor perfusion and blood vessel and cell membrane permeability that increased the drug supply to tumor cells. The effect of the pulsed MRgFUS treatment with PTX-loaded nanodroplets was clearly smaller than that of continuous wave MRgFUS treatment, supposedly due to significantly lower temperature increase as measured with MR thermometry and decreased extravasation. The time of the MRgFUS application after drug injection also proved to be an important factor with the best results observed when ultrasound was applied at least 6 h after the injection of drug-loaded nanodroplets. Some collateral damage was observed with particular ultrasound protocols supposedly associated with enhanced inflammation. CONCLUSION: This presented data suggest that there exists an optimal range of ultrasound application parameters and drug injection time. Decreased tumor growth, or complete resolution, was achieved with continuous wave ultrasound pressures below or equal to 3.1 MPa and drug injection times of at least 6 h prior to treatment. Increased acoustic pressure or ultrasound application before or shortly after drug injection gave increased tumor growth when compared to other protocols.

Phase-shift, stimuli-responsive perfluorocarbon nanodroplets for drug delivery to cancer.

This review focuses on phase-shift perfluorocarbon nanoemulsions whose action depends on an ultrasound-triggered phase shift from a liquid to gas state. For drug-loaded perfluorocarbon nanoemulsions, microbubbles are formed under the action of tumor-directed ultrasound and drug is released locally into tumor volume in this process. This review covers in detail mechanisms involved in the droplet-to-bubble transition as well as mechanisms of ultrasound-mediated drug delivery.

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.

Ultrasound-induced new cellular mechanism involved in drug resistance.

The acoustic effects in a biological milieu offer several scenarios for the reversal of multidrug resistance. In this study, we have observed higher sensitivity of doxorubicin-resistant uterine sarcoma MES-SA/DX5 cells to ultrasound exposure compared to its parent counterpart MES-SA cells; however, the results showed that the acoustic irradiation was genotoxic and could promote neotic division in exposed cells that was more pronounced in the resistant variant. The neotic progeny, imaged microscopically 24 hr post sonication, could contribute in modulating the final cell survival when an apoptotic dose of doxorubicin was combined with ultrasound applied either simultaneously or sequentially in dual-treatment protocols. Depending on the time and order of application of ultrasound and doxorubicin in combination treatments, there was either desensitization of the parent cells or sensitization of the resistant cells to doxorubicin action.

Phase-shift, stimuli-responsive drug carriers for targeted delivery.

The intersection of particles and directed energy is a rich source of novel and useful technology that is only recently being realized for medicine. One of the most promising applications is directed drug delivery. This review focuses on phase-shift nanoparticles (that is, particles of submicron size) as well as micron-scale particles whose action depends on an external-energy triggered, first-order phase shift from a liquid to gas state of either the particle itself or of the surrounding medium. These particles have tremendous potential for actively disrupting their environment for altering transport properties and unloading drugs. This review covers in detail ultrasound and laser-activated phase-shift nano- and micro-particles and their use in drug delivery. Phase-shift based drug-delivery mechanisms and competing technologies are discussed.

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.