Zebrafish Embryos Allow Prediction of Nanoparticle Circulation Times in Mice and Facilitate Quantification of Nanoparticle-Cell Interactions.

The zebrafish embryo is a vertebrate well suited for visualizing nanoparticles at high resolution in live animals. Its optical transparency and genetic versatility allow noninvasive, real-time observations of vascular flow of nanoparticles and their interactions with cells throughout the body. As a consequence, this system enables the acquisition of quantitative data that are difficult to obtain in rodents. Until now, a few studies using the zebrafish model have only described semiquantitative results on key nanoparticle parameters. Here, a MACRO dedicated to automated quantitative methods is described for analyzing important parameters of nanoparticle behavior, such as circulation time and interactions with key target cells, macrophages, and endothelial cells. Direct comparison of four nanoparticle (NP) formulations in zebrafish embryos and mice reveals that data obtained in zebrafish can be used to predict NPs' behavior in the mouse model. NPs having long or short blood circulation in rodents behave similarly in the zebrafish embryo, with low circulation times being a consequence of NP uptake into macrophages or endothelial cells. It is proposed that the zebrafish embryo has the potential to become an important intermediate screening system for nanoparticle research to bridge the gap between cell culture studies and preclinical rodent models such as the mouse.

Enhanced Permeability and Retention-like Extravasation of Nanoparticles from the Vasculature into Tuberculosis Granulomas in Zebrafish and Mouse Models.

The enhanced permeability and retention (EPR) effect is the only described mechanism enabling nanoparticles (NPs) flowing in blood to reach tumors by a passive targeting mechanism. Here, using the transparent zebrafish model infected with Mycobacterium marinum we show that an EPR-like process also occurs allowing different types of NPs to extravasate from the vasculature to reach granulomas that assemble during tuberculosis (TB) infection. PEGylated liposomes and other NP types cross endothelial barriers near infection sites within minutes after injection and accumulate close to granulomas. Although ∼100 and 190 nm NPs concentrated most in granulomas, even ∼700 nm liposomes reached these infection sites in significant numbers. We show by confocal microscopy that NPs can concentrate in small aggregates in foci on the luminal side of the endothelium adjacent to the granulomas. These spots are connected to larger foci of NPs on the ablumenal side of these blood vessels. EM analysis suggests that NPs cross the endothelium via the paracellular route. PEGylated NPs also accumulated efficiently in granulomas in a mouse model of TB infection with Mycobacterium tuberculosis, arguing that the zebrafish embryo model can be used to predict NP behavior in mammalian hosts. In earlier studies we and others showed that uptake of NPs by macrophages that are attracted to infection foci is one pathway for NPs to reach TB granulomas. This study reveals that when NPs are designed to avoid macrophage uptake, they can also efficiently target granulomas via an alternative mechanism that resembles EPR.

Anti-cancerous effect of albumin coated silver nanoparticles on MDA-MB 231 human breast cancer cell line.

With the aim of making specific targeting of silver nanoparticles as a drug for tumor cells and developing new anticancer agents, a novel nano-composite was developed. Albumin coated silver nanoparticles (ASNPs) were synthesized, and their anti-cancerous effects were evaluated against MDA-MB 231, a human breast cancer cell line. The synthesized ASNPs were characterized by spectroscopic methods. The morphological changes of the cells were observed by inverted, florescent microscopy and also by DNA ladder pattern on gel electrophoresis; the results revealed that the cell death process occurred through the apoptosis mechanism. It was found that ASNPs with a size of 90 nm and negatively charged with a zeta-potential of about -20 mV could be specifically taken up by tumor cells. The LD50 of ASNPs against MDA-MB 231 (5 µM), was found to be 30 times higher than that for white normal blood cells (152 µM). The characteristics of the synthesized ASNPs included; intact structure of coated albumin, higher cytotoxicity against cancer cells than over normal cells, and cell death based on apoptosis and reduction of gland tumor sizes in mice. This work indicates that ASNPs could be a good candidate for chemotherapeutic drug.

Zebrafish as a model system for characterization of nanoparticles against cancer.

Therapeutic nanoparticles (NPs) have great potential to deliver drugs against human diseases. Encapsulation of drugs in NPs protects them from being metabolized, while they are delivered specifically to a target site, thereby reducing toxicity and other side-effects. However, non-specific tissue accumulation of NPs, for example in macrophages, especially in the spleen and liver is a general problem with many NPs being developed for cancer therapy. To address the problem of non-specific tissue accumulation of NPs we describe the development of the zebrafish embryo as a transparent vertebrate system for characterization of NPs against cancer. We show that injection of human cancer cells results in tumor-like structures, and that subsequently injected fluorescent NPs, either made of polystyrene or liposomes can be imaged in real-time. NP biodistribution and general in vivo properties can be easily monitored in embryos having selective fluorescent labeling of specific tissues. We demonstrate in vitro, by using optical tweezer micromanipulation, microscopy and flow cytometry that polyethylene glycol (PEG) coating of NPs decreases the level of adhesion of NPs to macrophages, and also to cancer cells. In vivo in zebrafish embryos, PEG coating resulted in longer NP circulation times, decreased macrophage uptake, and reduced adhesion to the endothelium. Importantly, liposomes were observed to accumulate passively and selectively in tumor-like structures comprised of human cancer cells. These results show that zebrafish embryo is a powerful system for microscopy-based screening of NPs on the route to preclinical testing.

pH-responsive nano carriers for doxorubicin delivery.

PURPOSE: The aim of this study was to design stimuli-responsive nanocarriers for anti-cancer drug delivery. For this purpose, doxorubicin (DOX)-loaded, polysebacic anhydride (PSA) based nanocapsules (NC) were combined with pH-sensitive poly (L-histidine) (PLH). METHOD: PSA nano-carriers were first loaded with DOX and were coated with poly L-histidine to introduce pH sensitivity. The PLH-coated NCs were then covered with polyethylene glycol (PEG) to reduce macrophage uptake. The drug release profile from this system was examined in two different buffer solutions prepared as acidic (pH5) and physiological (pH 7.4) media. The physical and chemical properties of the nanocapsules were characterized by Fourier transform infrared spectroscopy (FTIR), dynamic light scattering (DLS), ultraviolet and visible absorption spectroscopy (UV-VIS), and scanning electron microscopy (SEM). In vitro studies of the prepared nanocapsules were conducted in MDA-MB-231 breast cancer cells. RESULTS: The results obtained by SEM and DLS revealed that nanocapsules have spherical morphology with an average size of 230 nm. Prepared pH sensitive nanocapsules exhibited pH-dependent drug release profile and promising intracellular release of drug. PEGylation of nanoparticles significantly prevented macrophage uptake compared to non-PEGylated particles.

Poly(sebacic anhydride) nanocapsules as carriers: effects of preparation parameters on properties and release of doxorubicin.

Poly(sebacic anhydride) (PSA) is a promising polymer for the production of drug delivery vehicles. The aim of this work is to study the effect of preparation parameters on the quality of the nanoparticles. In this study, doxorubicin (DOX)-loaded PSA nanocapsules were prepared by an emulsion method. Effects of factors such as type of organic solvent, co-solute (surfactant) and its concentration on drug-loading efficiency, particle size and size distribution, morphology and release profile were examined to gain insight in the preparation and stability of nanostructures. Particles with sizes in the range of 218-1198 nm were prepared. The smallest particles with a narrow size distribution were prepared by using polyvinyl alcohol as a co-solute and dichloromethane as a solvent. Efficiency and intracellular release of doxorubicin from the formulated particles were studied on MDA-MB-231 cells. It was observed that DOX-loaded PSA particles can diffuse into the cells and intracellular antitumour activity is directly related to the released amount of drug from the PSA nanocapsules.

Synthesis, characterization, and cellular uptake of magnetic nanocarriers for cancer drug delivery.

HYPOTHESIS: The absence of targetability is the primary inadequacy of conventional chemotherapy. Targeted drug delivery systems are conceptualized to overcome this challenge. We have designed a targetable magnetic nanocarrier consisting of a superparamagnetic iron oxide (SPIO) core and biocompatible and biodegradable poly(sebacic anhydride)-block-methyl ether poly(ethylene glycol) (PSA-mPEG) polymer shell. The idea is that this type of carriers should facilitate the targeting of cancer cells. EXPERIMENTS: PSA-mPEG was synthesized with poly-condensation and the in vitro degradation rate of the polymer was monitored by gel permeation chromatography (GPC). The magnetic nanocarriers were fabricated devoid of any surfactants and were capable of carrying high payload of hydrophobic dye. The successful encapsulation of SPIO within the polymer shell was confirmed by TEM. The results we obtained from measuring the size of SPIO loaded in polymeric NPs (SPIO-PNP) by dynamic light scattering (DLS) and iron content measurement of these particles by ICP-MS, indicate that SPIO is the most suitable carrier for cancer drug delivery applications. FINDINGS: Measuring the hydrodynamic radii of SPIO-PNPs by DLS over one month revealed the high stability of these particles at both body and room temperature. We further investigated the cell viability and cellular uptake of SPIO-PNPs in vitro with MDA-MB-231 breast cancer cells. We found that SPIO-PNPs induce negligible toxicity within a concentration range of 1-2µg/ml. The TEM micrographs of thin cross-sectioned MDA-MBA-231 cells showed internalization of SPIO-PNPs within size range of 150-200nm after 24h. This study has provided a foundation for eventually loading these nanoparticles with anti-cancer drugs for targeted cancer therapy using an external magnetic field.