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.
Background Drug delivery in polymeric micelles combined with tumor irradiation by ultrasound results in effective drug targeting, but this technique requires prior tumor imaging. A technology that ...combined ultrasound imaging with ultrasound-mediated nanoparticle-based targeted chemotherapy could therefore have important applications in cancer treatment. Methods Mixtures of drug-loaded polymeric micelles and perfluoropentane (PFP) nano/microbubbles stabilized by the same biodegradable block copolymer were prepared. Size distribution of nanoparticles was measured by dynamic light scattering. Cavitation activity (oscillation, growth, and collapse of microbubbles) under ultrasound was assessed based on the changes in micelle/microbubble volume ratios. The effect of the nano/microbubbles on the ultrasound-mediated cellular uptake of doxorubicin (Dox) in MDA MB231 breast tumors in vitro and in vivo (in mice bearing xenograft tumors) was determined by flow cytometry. Statistical tests were two-sided. Results Phase state and nanoparticle sizes were sensitive to the copolymer/perfluorocarbon volume ratio. At physiologic temperatures, nanodroplets converted into nano/microbubbles. Doxorubicin was localized in the microbubble walls formed by the block copolymer. Upon intravenous injection into mice, Dox-loaded micelles and nanobubbles extravasated selectively into the tumor interstitium, where the nanobubbles coalesced to produce microbubbles with a strong, durable ultrasound contrast. Doxorubicin was strongly retained in the microbubbles but released in response to therapeutic ultrasound. Microbubbles cavitated under the action of tumor-directed ultrasound, which enhanced intracellular Dox uptake by tumor cells in vitro to a statistically significant extent relative to that observed with unsonicated microbubbles (drug uptake ratio = 4.60; 95% confidence interval CI = 1.70 to 12.47; P = .017) and unsonicated micelles (drug uptake ratio = 7.97; 95% CI = 3.72 to 17.08; P = .0032) and resulted in tumor regression in the mouse model. Conclusions Multifunctional nanoparticles that are tumor-targeted drug carriers, long-lasting ultrasound contrast agents, and enhancers of ultrasound-mediated drug delivery have been developed and deserve further exploration as cancer therapeutics.
The paper reports the results of nanotherapy of ovarian, breast, and pancreatic cancerous tumors by paclitaxel-loaded nanoemulsions that convert into microbubbles locally in tumor tissue under the ...action of tumor-directed therapeutic ultrasound. Tumor accumulation of nanoemulsions was confirmed by ultrasound imaging. Dramatic regression of ovarian, breast, and orthotopic pancreatic tumors was observed in tumor therapy through systemic injections of drug-loaded nanoemulsions combined with therapeutic ultrasound, signifying efficient ultrasound-triggered drug release from tumor-accumulated nanodroplets. The mechanism of drug release in the process of droplet-to-bubble conversion is discussed. No therapeutic effect from the nanodroplet/ultrasound combination was observed without the drug, indicating that therapeutic effect was caused by the ultrasound-enhanced chemotherapeutic action of the tumor-targeted drug, rather than the mechanical or thermal action of ultrasound itself. Tumor recurrence was observed after the completion of the first treatment round; a second treatment round with the same regimen proved less effective, suggesting that drug-resistant cells were either developed or selected during the first treatment round.
Schematic illustration of drug transfer from nanodroplets into cells under the action of ultrasound (up) and the photographs of the ovarian cancer bearing mouse at various stages of treatment (down).
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A new class of multifunctional nanoparticles that combine properties of polymeric drug carriers, ultrasound imaging contrast agents, and enhancers of ultrasound-mediated drug delivery has been ...developed. At room temperature, the developed systems comprise perfluorocarbon nanodroplets stabilized by the walls made of biodegradable block copolymers. Upon heating to physiological temperatures, the nanodroplets convert into nano/microbubbles. The phase state of the systems and bubble size may be controlled by the copolymer/perfluorocarbon volume ratio. Upon intravenous injections, a long-lasting, strong and selective ultrasound contrast is observed in the tumor volume indicating nanobubble extravasation through the defective tumor microvasculature, suggesting their coalescence into larger, highly echogenic microbubbles in the tumor tissue. Under the action of tumor-directed ultrasound, microbubbles cavitate and collapse resulting in a release of the encapsulated drug and dramatically enhanced intracellular drug uptake by the tumor cells. This effect is tumor-selective; no accumulation of echogenic microbubbles is observed in other organs. Effective chemotherapy of the MDA MB231 breast cancer tumors has been achieved using this technique.
Perfluorocarbon nanoemulsions can deliver lipophilic therapeutic agents to solid tumors and simultaneously provide for monitoring nanocarrier biodistribution via ultrasonography and/or 19F 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 (19F) MR contrast properties, which allows using multimodal imaging and 19F 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 19F 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.
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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.
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. WIREs Nanomed Nanobiotechnol 2012, 4:492–510. doi: 10.1002/wnan.1176
This article is categorized under:
Therapeutic Approaches and Drug Discovery > Emerging Technologies
Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease
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.
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Block copolymer micelles are generally formed by self-assembly of amphiphilic copolymer molecules in an aqueous milieu. The hydrophobic blocks (or polyion complexes) form the micelle cores while the ...hydrophilic blocks form the micelle corona (or shells). Consequently, lipophilic drugs are solubilized in the hydrophobic micelle cores, which dramatically increases drug concentration in an aqueous environment. This offers new life to bioactive compounds abandoned due to low aqueous solubility. Polymeric micelles can be designed to avoid extravasation to normal tissues and recognition by the reticulo-endothelial system cells; this prolongs their circulation time after systemic injection. This in turn provides passive targeting to cancerous or inflamed tissues via the enhanced permeability and retention effect. Active tumor-targeting may be achieved by modifying the micelle surface with specific ligands to tumor cell receptors or creating “immunomicelles” by attaching monoclonal antibodies to specific antigens over-expressed on the cancerous cell surface. A different approach to active tumor targeting consists of developing stimuli-responsive micelles that release their drug load only in response to environmental or physical stimuli, such as the lower pH in tumor tissue, heat, sound, or light. Currently, a number of polymeric micelles are in various phases of pre-clinical and clinical development. Present paper reviews recent advances in the development of physical stimuli-responsive micelles with high targeting potentials and controlled on-demand drug release.
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.
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