Liron Limor Israel

Biocompatible nanocomposite for PET/MRI hybrid imaging

A novel nanocarrier system was designed and developed with key components uniquely structured at the nanoscale for early cancer diagnosis and treatment. In order to perform magnetic resonance imaging, hydrophilic superparamagnetic maghemite nanoparticles (NPs) were synthesized and coated with a lipophilic organic ligand. Next, they were entrapped into polymeric NPs made of biodegradable poly(lactic-co-glycolic acid) linked to polyethylene glycol. In addition, resulting NPs have been conjugated on their surface with a 2,2′-(7-(4-((2-aminoethyl)amino)-1-carboxy-4-oxobutyl)-1,4,7-triazonane-1,4-diyl)diacetic acid ligand for subsequent 68Ga incorporation. A cell-based cytotoxicity assay has been employed to verify the in vitro cell viability of human pancreatic cancer cells exposed to this nanosystem. Finally, in vivo positron emission tomography-computerized tomography biodistribution studies in healthy animals were performed.

miR-142 orchestrates a network of actin cytoskeleton regulators during megakaryopoiesis

Genome-encoded microRNAs (miRNAs) provide a posttranscriptional regulatory layer that controls the differentiation and function of various cellular systems, including hematopoietic cells. miR-142 is one of the most prevalently expressed miRNAs within the hematopoietic lineage. To address the in vivo functions of miR-142, we utilized a novel reporter and a loss-of-function mouse allele that we have recently generated. In this study, we show that miR-142 is broadly expressed in the adult hematopoietic system. Our data further reveal that miR-142 is critical for megakaryopoiesis. Genetic ablation of miR-142 caused impaired megakaryocyte maturation, inhibition of polyploidization, abnormal proplatelet formation, and thrombocytopenia. Finally, we characterized a network of miR-142-3p targets which collectively control actin filament homeostasis, thereby ensuring proper execution of actin-dependent proplatelet formation. Our study reveals a pivotal role for miR-142 activity in megakaryocyte maturation and function, and demonstrates a critical contribution of a single miRNA in orchestrating cytoskeletal dynamics and normal hemostasis. DOI: http://dx.doi.org/10.7554/eLife.01964.001

Targeted diagnostic magnetic nanoparticles for medical imaging of pancreatic cancer.

Highly aggressive cancer types such as pancreatic cancer possess a mortality rate of up to 80% within the first 6months after diagnosis. To reduce this high mortality rate, more sensitive diagnostic tools allowing an early stage medical imaging of even very small tumours are needed. For this purpose, magnetic, biodegradable nanoparticles prepared using recombinant human serum albumin (rHSA) and incorporated iron oxide (maghemite, γ-Fe2O3) nanoparticles were developed. Galectin-1 has been chosen as target receptor as this protein is upregulated in pancreatic cancer and its precursor lesions but not in healthy pancreatic tissue nor in pancreatitis. Tissue plasminogen activator derived peptides (t-PA-ligands), that have a high affinity to galectin-1 have been chosen as target moieties and were covalently attached onto the nanoparticle surface. Improved targeting and imaging properties were shown in mice using single photon emission computed tomography-computer tomography (SPECT-CT), a handheld gamma camera, and magnetic resonance imaging (MRI).

Intradermal air pouch leukocytosis as an in vivo test for nanoparticles

The need for test systems for nanoparticle biocompatibility, toxicity, and inflammatory or adaptive immunological responses is paramount. Nanoparticles should be free of microbiological and chemical contaminants, and devoid of toxicity. Nevertheless, in the absence of contamination, these particles may still induce undesired immunological effects in vivo, such as enhanced autoimmunity, hypersensitivity reactions, and fibrosis. Here we show that artificial particles of specific sizes affect immune cell recruitment as tested in a dermal air pouch model in mice. In addition, we demonstrate that the composition of nanoparticles may influence immune cell recruitment in vivo. Aside from biophysical characterizations in terms of hydrodynamic diameter, zeta potential, concentration, and atomic concentration of metals, we show that – after first-line in vitro assays – characterization of cellular and molecular effects by dermal air pouch analysis is straightforward and should be included in the quality control of nanoparticles. We demonstrate this for innate immunological effects such as neutrophil recruitment and the production of immune-modulating matrix metalloproteases such as MMP-9; we propose the use of air pouch leukocytosis analysis as a future standard assay.

Physico-chemical and toxicological characterization of iron-containing albumin nanoparticles as platforms for medical imaging

Iron oxide-containing magnetic nanoparticles (MNPs) have certain advantages over currently used contrast agents for tumor imaging by magnetic resonance imaging (MRI) as they offer the possibility of functionalization with ligands and tracers. Functionalized MNPs also may be used for targeted tumor therapy. In the current study nanoparticles (NPs) consisting of recombinant human serum albumin (rHSA) with incorporated hydrophilic (NH4)2Ce(IV)(NO3)6-γ-Fe2O3 particles (CAN maghemite particles) for medical imaging were produced and characterized. For this purpose CAN maghemite particles were incorporated into an rHSA matrix to yield rHSA-NPs. The resulting NPs were analyzed by transmission electron microscopy, photon correlation spectroscopy, and atomic absorption. The sizes of the manufactured NP were 170 ± 10 nm, and the zeta-potential was -50 ± 3 mV. The NPs remained stable when stored after lyophilization with sucrose 3% [w/v] as a cryoprotector. They showed pro-inflammatory properties without cell and animal toxicity in vivo and were highly contrasting in MRI. In conclusion, this report introduces novel rHSA NP with favorable properties containing iron oxide for detection by MRI.

Matrix metalloproteinase-9 (MMP-9) as an activator of nanosystems for targeted drug delivery in pancreatic cancer

Specific cancer cell targeting is a pre-requisite for efficient drug delivery as well as for high-resolution imaging and still represents a major technical challenge. Tumor-associated enzyme-assisted targeting is a new concept that takes advantage of the presence of a specific activity in the tumor entity. MMP-9 is a protease found to be upregulated in virtually all malignant tumors. Consequently, we hypothesized that its presence can provide a de-shielding activity for targeted delivery of drugs by nanoparticles (NPs) in pancreatic cancer. Here, we describe synthesis and characterization of an optimized MMP-9-cleavable linker mediating specific removal of a PEG shield from a PLGA-b-PEG-based polymeric nanocarrier (Magh@PNPs-PEG-RegaCP-PEG) leading to specific uptake of the smaller PNPs with their cargo into cells. The specific MMP-9-cleavable linker was designed based on the degradation efficiency of peptides derived from the collagen type II sequence. MMP-9-dependent uptake of the Magh@PNPs-PEG-RegaCP-PEG was demonstrated in pancreatic cancer cells in vitro. Accumulation of the Magh@PNPs-PEG-RegaCP-PEG in pancreatic tissues in the clinically relevant KPC mouse model of pancreatic cancer, as a proof-of-concept, was tumor-specific and MMP-9-dependent, indicating that MMP-9 has a strong potential as a specific mediator of PNP de-shielding for tumor-specific uptake. Pre-treatment of mice with Magh@PNPs-PEG-RegaCP-PEG led to reduction of liver metastasis and drastically decreased average colony size. In conclusion, the increased tumor-specific presence and activity of MMP-9 can be exploited to deliver an MMP-9-activatable NP to pancreatic tumors specifically, effectively, and safely.

Ultrasound-Mediated Surface Engineering of Theranostic Magnetic Nanoparticles: An Effective One-Pot Functionalization Process UsingMixed Polymers for siRNA Delivery

Nano-sized materials have been studied for diverse clinical applications, partly because their size-dependent physical properties and nanometer-scale dimensions have important roles in biological systems. Synergistic combinations of differently nanostructured materials, such as polymer-coated magnetic nanoparticles (NPs), strongly promoted various multifunctional nano-medical platforms for simultaneous diagnosis and therapy in the rapidly emerging area of theranostics. In this context, magnetically responsive Ce cation-doped maghemite (γ-Fe2O3) NPs form a useful NPs basis towards a new type of polycationic polymer/organic species-grafted maghemite NPs for both drug delivery and imaging. The versatility of the Ce cation-doped maghemite NPs fabrication process mediated by high-power ultrasound (US) enables the development of a new one-step time-saving US-driven variant fabrication of corresponding polymer/organic species-grafted NPs. Thus, two types of organic polycationic species, a branched 25 kDa polyethylene imine (b-PEI25) polymer and a generation 2 (G2) PAMAM (poly(amidoamine)) dendrimer biopolymer, were simultaneously used during this US-mediated NPs system fabrication to effectively deliver optimized small interfering RNA (siRNA) applications as a proof of concept. This unique one-step fabrication protocol affords a positively charged magnetic core grafted with mixed organic species nanocomposite particles that enables both gene silencing therapy and magnetic resonance imaging.

MagRET Nanoparticles: An Iron Oxide Nanocomposite Platform for Gene Silencing from MicroRNAs to Long Noncoding RNAs

Silencing of RNA to knock down genes is currently one of the top priorities in gene therapies for cancer. However, to become practical the obstacle of RNA delivery needs to be solved. In this study, we used innovative maghemite (γ-Fe2O3) nanoparticles, termed magnetic reagent for efficient transfection (MagRET), which are composed of a maghemite core that is surface-doped by lanthanide Ce(3/4+) cations using sonochemistry. Thereafter, a polycationic polyethylenimine (PEI) polymer phase is bound to the maghemite core via coordinative chemistry enabled by the [CeL(n)](3/4+)cations/complex. PEI oxidation was used to mitigate the in vivo toxicity. Using this approach, silencing of 80-100% was observed for mRNAs, microRNAs, and lncRNA in a variety of cancer cells. MagRET NPs are advantageous in hard to transfect leukemias. This versatile nanoscale carrier can silence all known types of RNAs and these MagRET NPs with oxidized PEI are not lethal upon injection, thus holding promise for therapeutic applications, as a theranostic tool.

Development of Multifunctional Magnetic Nanoparticles for Genetic Engineering and Tracking of Neural Stem Cells

Genetic modification of cell transplant populations and cell tracking ability are key underpinnings for effective cell therapies. Current strategies to achieve these goals utilize methods which are unsuitable for clinical translation because of related safety issues, and multiple protocol steps adding to cost and complexity. Multifunctional magnetic nanoparticles (MNPs) offering dual mode gene delivery and imaging contrast capacity offer a valuable tool in this context. Despite their key benefits, there is a critical lack of neurocompatible and multifunctional particles described for use with transplant populations for neurological applications. Here, a systematic screen of MNPs (using a core shown to cause contrast in magnetic resonance imaging (MRI)) bearing various surface chemistries (polyethylenimine (PEI) and oxidized PEI and hybrids of oxidized PEI/alginic acid, PEI/chitosan and PEI/polyamidoamine) is performed to test their ability to genetically engineer neural stem cells (NSCs; a cell population of high clinical relevance for central nervous system disorders). It is demonstrated that gene delivery to NSCs can be safely achieved using two of the developed formulations (PEI and oxPEI/alginic acid) when used in conjunction with oscillating magnetofection technology. After transfection, intracellular particles can be detected by histological procedures with labeled cells displaying contrast in MRI (for real time cell tracking).

Acute in Vivo Toxicity Mitigation of PEI-Coated Maghemite Nanoparticles Using Controlled Oxidation and Surface Modifications toward siRNA Delivery

A ceric ammonium nitrate (CAN)-based doping step was used for the fabrication of core maghemite nanoparticles (NPs) that enabled the obtainment of colloid particles with a view to a high-level nanoparticle (NP) surface doping by Ce(III/IV). Such doping of Ce(III/IV) cations enables one to exploit their quite rich coordination chemistry for ligand coordinative binding. In fact, they were shown to act as powerful Lewis acid centers for attaching any organic (Lewis base) ligand such as a 25 kDa branched PEI polymer. Resulting conPEI25-CAN-γ-Fe2O3 NPs have been fully characterized before a successful implementation of siRNA loading and cell delivery/gene silencing using a well-known dual luciferase system. This attractive result emphasized their significant potential as an NP platform technology toward additional MRI and/or drug delivery (peptide)-relating end applications. However, due to their high positive charge, PEI polymers can cause severe in vivo toxicity due to their interaction with negatively charged red blood cells (RBC), resulting in RBC aggregation and lysis, leading to thrombosis and, finally, to animal death. In order to mitigate these acute toxic effects, two different types of surface modifications were performed. One modification included the controlled oxidation of 0.1-5% of the PEI amines before or after conjugation to the NPs, using hydrogen peroxide or potassium persulfate. The other type of modification was the addition of a second biocompatible polyanionic polymer to the PEI grafted NPs, based on the concept of a layer-by-layer (LbL) technique. This modification is based on the coordination of another polyanionic polymer on the NPs surface in order to create a combined hybrid PEI and polyanionic polymer nanosystem. In both cases, the surface modification successfully mitigated the NP acute in vivo toxicity, without compromising the silencing efficiency.