Recent advances in ultrasound-based diagnosis and therapy with micro- and nanometer-sized formulations.

Ultrasound (US) is one of the most frequently used imaging methods in the clinic. The broad spectrum of its applications can be increased by the use of gas-filled microbubbles (MB) as ultrasound contrast agents (UCA). In recent years, also nanoscale UCA like nanobubbles (NB), echogenic liposomes (ELIP) and nanodroplets have been developed, which in contrast to MB, are able to extravasate from the vessels into the tissue. New disease-specific UCA have been designed for the assessment of tissue biomarkers and advanced US to a molecular imaging modality. For this purpose, specific binding moieties were coupled to the UCA surface. The vascular endothelial growth factor receptor-2 (VEGFR-2) and P-/E-selectin are prominent examples of molecular US targets to visualize tumor blood vessels and inflammatory diseases, respectively. Besides their application in contrast-enhanced imaging, MB can also be employed for drug delivery to tumors and across the blood-brain barrier (BBB). This review summarizes the development of micro- and nanoscaled UCA and highlights recent advances in diagnostic and therapeutic applications, which are ready for translation into the clinic.

Micro-computed tomography (µCT) as a novel method in ecotoxicology--determination of morphometric and somatic data in rainbow trout (Oncorhynchus mykiss).

Fish are important sentinel organisms for the assessment of water quality and play a central role in ecotoxicological research. Of particular importance to the assessment of health and fitness of fish stocks in response to environmental conditions or pollution are morphometric (e.g. Fulton's condition index) and somatic indices (e.g. hepatosomatic, and gonadosomatic index). Standard measurements of somatic indices are invasive and require, by definition, the sacrifice of examined animals, thus prohibiting longitudinal studies and relocation of animals captured in the field. As a potential solution, in the present study, we propose the use of micro-computed tomography (µCT) as imaging modality to non-invasively tomographically image rainbow trout (Oncorhynchus mykiss) exposed to different sediment suspensions. We here demonstrate that µCT can be used as a tool to reliably measure the volumes of different organs, which could then be applied as a substitute of their weights in calculation of somatic indices. To the best of our knowledge, this study is the first to report the results of µCT analyses in the context of ecotoxicological research in rainbow trout. It has the potential to greatly increase the information value of experiments conducted with fish and also to potentially reduce the number of animals required for studying temporal effects through facilitating longitudinal studies within the same individuals.

Liposomal delivery of dexamethasone attenuates prostate cancer bone metastatic tumor growth in vivo.

BACKGROUND: The inflammatory tumor microenvironment, and more specifically the tumor-associated macrophages, plays an essential role in the development and progression of prostate cancer towards metastatic bone disease. Tumors are often characterized by a leaky vasculature, which - combined with the prolonged circulation kinetics of liposomes - leads to efficient tumor localization of these drug carriers, via the so-called enhanced permeability and retention (EPR) -effect. In this study, we evaluated the utility of targeted, liposomal drug delivery of the glucocorticoid dexamethasone in a model of prostate cancer bone metastases. METHODS: Tumor-bearing Balb-c nu/nu mice were treated intravenously with 0.2-1.0-5.0 mg/kg/week free- and liposomal DEX for 3-4 weeks and tumor growth was monitored by bioluminescent imaging. RESULTS: Intravenously administered liposomes localize efficiently to bone metastases in vivo and treatment of established bone metastases with (liposomal) dexamethasone resulted in a significant inhibition of tumor growth up to 26 days after initiation of treatment. Furthermore, 1.0 mg/kg liposomal dexamethasone significantly outperformed 1.0 mg/kg free dexamethasone, and was found to be well-tolerated at clinically-relevant dosages that display potent anti-tumor efficacy. CONCLUSIONS: Liposomal delivery of the glucocorticoid dexamethasone inhibits the growth of malignant bone lesions. We believe that liposomal encapsulation of dexamethasone offers a promising new treatment option for advanced, metastatic prostate cancer which supports further clinical evaluation.

Fluorescent cell-traceable dexamethasone-loaded liposomes for the treatment of inflammatory liver diseases.

Liposomes are routinely used carrier materials for delivering drug molecules to pathological sites. Besides in tumors and inflammatory sites, liposomes also strongly accumulate in liver and spleen. The potential of using liposomes to treat acute and chronic liver disorders, however, has not yet been evaluated. We here explored the therapeutic potential of dexamethasone (Dex)-loaded liposomes for inflammatory liver diseases, using experimental models of acute and chronic liver injury in mice. Fluorescently labeled liposomes predominantly accumulated in hepatic phagocytes, but also in T cells. Importantly, Dex-loaded liposomes reduced T cells in blood and liver, more effectively than free Dex, and endorsed the anti-inflammatory polarization of hepatic macrophages. In experimental chronic liver damage, Dex-loaded liposomes significantly reduced liver injury and liver fibrosis. In immune-mediated acute hepatitis Dex-loaded liposomes, but not free Dex, significantly reduced disease severity. T cells, not macrophages, were significantly depleted by Dex liposomes in liver disease models in vivo, as further supported by mechanistic cell death in vitro studies. Our data indicate that Dex liposomes may be an interesting treatment option for liver diseases, in particular for immune-mediated hepatitis. The depletion of T cells might represent the major mechanism of action of Dex liposomes, rather than their macrophage-polarizing activities.

Decationized polyplexes as stable and safe carrier systems for improved biodistribution in systemic gene therapy.

Many polycation-based gene delivery vectors show high transfection in vitro, but their cationic nature generally leads to significant toxicity and poor in vivo performance which significantly hampers their clinical applicability. Unlike conventional polycation-based systems, decationized polyplexes are based on hydrophilic and neutral polymers. They are obtained by a 3-step process: charge-driven condensation followed by disulfide crosslinking stabilization and finally polyplex decationization. They consist of a disulfide-crosslinked poly(hydroxypropyl methacrylamide) (pHPMA) core stably entrapping plasmid DNA (pDNA), surrounded by a shell of poly(ethylene glycol) (PEG). In the present paper the applicability of decationized polyplexes for systemic administration was evaluated. Cy5-labeled decationized polyplexes were evaluated for stability in plasma by fluorescence single particle tracking (fSPT), which technique showed stable size distribution for 48 h unlike its cationic counterpart. Upon the incubation of the polymers used for the formation of polyplexes with HUVEC cells, MTT assay showed excellent cytocompatibility of the neutral polymers. The safety was further demonstrated by a remarkable low teratogenicity and mortality activity of the polymers in a zebrafish assay, in great contrast with their cationic counterpart. Near infrared (NIR) dye-labeled polyplexes were evaluated for biodistribution and tumor accumulation by noninvasive optical imaging when administered systemically in tumor bearing mice. Decationized polyplexes exhibited an increased circulation time and higher tumor accumulation, when compared to their cationic precursors. Histology of tumors sections showed that decationized polyplexes induced reporter transgene expression in vivo. In conclusion, decationized polyplexes are a platform for safer polymeric vectors with improved biodistribution properties when systemically administered.

The Theranostic Path to Personalized Nanomedicine.

Advances in nanotechnology and chemical engineering have led to the development of many different drug delivery systems. These 1-100(0) nm-sized carrier materials aim to increase drug concentrations at the pathological site, while avoiding their accumulation in healthy non-target tissues, thereby improving the balance between the efficacy and the toxicity of systemic (chemo-) therapeutic interventions. An important advantage of such nanocarrier materials is the ease of incorporating both diagnostic and therapeutic entities within a single formulation, enabling them to be used for theranostic purposes. We here describe the basic principles of using nanomaterials for targeting therapeutic and diagnostic agents to pathological sites, and we discuss how nanotheranostics and image-guided drug delivery can be used to personalize nanomedicine treatments.

In Vivo Nanotoxicity Testing using the Zebrafish Embryo Assay.

Nanoparticles are increasingly used for biomedical purposes. Many different diagnostic and therapeutic applications are envisioned for nanoparticles, but there are often also serious concerns regarding their safety. Given the fact that numerous new nanomaterials are being developed every day, and that not much is known about the long-term toxicological impact of exposure to nanoparticles, there is an urgent need to establish efficient methods for nanotoxicity testing. The zebrafish (Danio rerio) embryo assay has recently emerged as an interesting 'intermediate' method for in vivo nanotoxicity screening, enabling (semi-) high-throughput analyses in a system significantly more complex than cultured cells, but at the same time also less 'invasive' and less expensive than large-scale biocompatibility studies in mice or rats. The zebrafish embryo assay is relatively well-established in the environmental sciences, but it has not yet gained wide notice in the nanomedicine field. Using prototypic polymeric drug carriers, gold-based nanodiagnostics and nanotherapeutics, and iron oxide-based nanodiagnostics, we here show that toxicity testing using zebrafish embryos is easy, efficient and informative, and faithfully reflects, yet significantly extends, cell-based toxicity testing. We therefore expect that the zebrafish embryo assay will become a popular future tool for in vivo nanotoxicity screening.

Recent progress in nanomedicine: therapeutic, diagnostic and theranostic applications.

In recent years, the use of nanomedicine formulations for therapeutic and diagnostic applications has increased exponentially. Many different systems and strategies have been developed for drug targeting to pathological sites, as well as for visualizing and quantifying important (patho-) physiological processes. In addition, ever more efforts have been undertaken to combine diagnostic and therapeutic properties within a single nanomedicine formulation. These so-called nanotheranostics are able to provide valuable information on drug delivery, drug release and drug efficacy, and they are considered to be highly useful for personalizing nanomedicine-based (chemo-) therapeutic interventions.

Personalized nanomedicine.

Personalized medicine aims to individualize chemotherapeutic interventions on the basis of ex vivo and in vivo information on patient- and disease-specific characteristics. By noninvasively visualizing how well image-guided nanomedicines-that is, submicrometer-sized drug delivery systems containing both drugs and imaging agents within a single formulation, and designed to more specifically deliver drug molecules to pathologic sites-accumulate at the target site, patients likely to respond to nanomedicine-based therapeutic interventions may be preselected. In addition, by longitudinally monitoring how well patients respond to nanomedicine-based therapeutic interventions, drug doses and treatment protocols can be individualized and optimized during follow-up. Furthermore, noninvasive imaging information on the accumulation of nanomedicine formulations in potentially endangered healthy tissues may be used to exclude patients from further treatment. Consequently, combining noninvasive imaging with tumor-targeted drug delivery seems to hold significant potential for personalizing nanomedicine-based chemotherapeutic interventions, to achieve delivery of the right drug to the right location in the right patient at the right time.