Yael Levi-Kalisman

Delivery of Liposomal Quantum Dots via Monocytes for Imaging of Inflamed Tissue

Quantum dots (QDs), semiconductor nanocrystals, are fluorescent nanoparticles of growing interest as an imaging tool of a diseased tissue. However, a major concern is their biocompatibility, cytotoxicity, and fluorescence instability in biological milieu, impeding their use in biomedical applications, in general, and for inflammation imaging, in particular. In addition, for an efficient fluorescent signal at the desired tissue, and avoiding systemic biodistribution and possible toxicity, targeting is desired. We hypothesized that phagocytic cells of the innate immunity system (mainly circulating monocytes) can be exploited as transporters of specially designed liposomes containing QDs to the inflamed tissue. We developed a liposomal delivery system of QDs (LipQDs) characterized with high encapsulation yield, enhanced optical properties including far-red emission wavelength and fluorescent stability, high quantum yield, and protracted fluorescent decay lifetime. Treatment with LipQDs, rather than free QDs, exhibited high accumulation and retention following intravenous administration in carotid-injured rats (an inflammatory model). QD-monocyte colocalization was detected in the inflamed arterial segment only following treatment with LipQDs. No cytotoxicity was observed following LipQD treatment in cell cultures, and changes in liver enzymes and gross histopathological changes were not detected in mice and rats, respectively. Our results suggest that the LipQD formulation could be a promising strategy for imaging inflammation.

Synthesis and Properties of Novel Silver-Containing DNA Molecules

Migration of silver atoms from silver nano-particles selectively to a double-stranded poly(dG)-poly(dC) polymer leads to metallization of the DNA. As a result the DNA molecules become shorter and thicker (higher), as evident from the atomic force microscopy imaging analysis. The metalized molecules can be detected by transmission and scanning electron microscopy in contrast to the initial non-metalized ones.

liposomal temozolomide drug delivery using convection enhanced delivery

Abstract Even though some progress in diagnosis and treatment has been made over the years, there is still no definitive treatment available for Glioblastoma multiforme (GBM). Convection‐enhanced delivery (CED), a continuous infusion‐mediated pressure gradient via intracranial catheters, studied in clinical trials, enables in situ drug concentrations several orders of magnitude greater than those achieved by systemic administration. We hypothesized that the currently limited efficacy of CED could be enhanced by a liposomal formulation, thus achieving enhanced drug localization to the tumor site with minimal toxicity. We hereby describe a novel approach for treating GBM by CED of liposomes containing the known chemotherapeutic agent, temozolomide (TMZ). A new technique for encapsulating TMZ in hydrophilic (PEGylated) liposomes, characterized by nano‐size (121 nm), low polydispersity index (<0.13) and with near‐neutral charge (−3,0.2 mV), has been developed. Co‐infusion of PEGylated Gd‐DTPA liposomes and TMZ‐liposomes by CED in GBM bearing rats, resulted in enhanced tumor detection with longer residence time than free Gd‐DTPA. Treatment of GBM‐bearing rats with either TMZ solution or TMZ‐liposomes resulted in greater tumor inhibition and significantly higher survival. However, the longer survival and smaller tumor volumes exhibited by TMZ liposomal treatment in comparison to TMZ in solution were insignificant (p < 0.053); and only significantly lower edema volumes were observed. Thus, there are no clear‐cut advantages to use a liposomal delivery system of TMZ via CED over a drug solution. Graphical abstract Figure. No Caption available.

pH and Salt Effects on Surface Activity and Self-Assembly of Copolymers Containing a Weak Polybase

Copolymers with well-defined architectures, controlled molecular weights, and narrow molar mass dispersities (Đ) were synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization. The resultant polymers contain different combinations of the pH-responsive monomer 2-(diethylaminoethyl) methacrylate (DEAEMA), the hydrophobic comonomer butyl methacrylate (BMA), and a neutral hydrophilic stabilizing monomer polyethylene glycol monomethyl ether methacrylate (designated O950). Surface tension and cryo-TEM measurements of native and heavy-atom stained samples were used to characterize the pH and salt responsiveness of the different polymers as a function of their composition. These studies indicate that while the polymers predominately self-assemble to form spherical micelles, a narrow size distribution is observed in aqueous solutions of poly(O950)-b-(BMA) and poly(O950)-b-(DEAEMA-co-BMA), whereas a broad size distribution characterizes the assemblies of poly(O950)-b-(DEAEMA) and poly(DEAEMA-co-BMA). In the latter case, micelles having diameters around 15-25 nm are found along with smaller aggregates (about 10 nm) mostly arranged in elongated necklace-like structures. The pH and salt-responsiveness of the DEAEMA residue, as indicated by the surface activity of the copolymers, was found to depend on the nature of the additional components: covalently linked hydrophobic groups (BMA) moderated the pH response of the copolymer as compared to nonionic and hydrophilic groups as in poly(O950)-b-(DEAEMA). These results suggest that mutual interactions among the building blocks of self-assembling copolymers should be taken into account when designing responsive copolymers.

Concentration-based self-assembly of phycocyanin

Cyanobacteria light-harvesting complexes can change their structure to cope with fluctuating environmental conditions. Studying in vivo structural changes is difficult owing to complexities imposed by the cellular environment. Mimicking this system in vitro is challenging, as well. The in vivo system is highly concentrated, and handling similar in vitro concentrated samples optically is difficult because of high absorption. In this research, we mapped the cyanobacteria antennas self-assembly pathways using highly concentrated solutions of phycocyanin (PC) that mimic the in vivo condition. PC was isolated from the thermophilic cyanobacterium Thermosynechococcus vulcanus and measured by several methods. PC has three oligomeric states: hexamer, trimer, and monomer. We showed that the oligomeric state was changed upon increase of PC solution concentration. This oligomerization mechanism may enable photosynthetic organisms to adapt their light-harvesting system to a wide range of environmental conditions.

Surface Charge Influence on the Phase Separation and Viscosity of Cellulose Nanocrystals

A series of four cellulose nanocrystal (CNC) suspensions were prepared from bleached softwood kraft pulp using different conditions of sulfuric acid hydrolysis. The CNCs were identical in size (95 nm in length × 5 nm in width) but had different surface charges corresponding to the harshness of the hydrolysis conditions. Consequently, it was possible to isolate the effects of surface charge on the self-assembly and viscosity of the CNC suspensions across surface charges ranging from 0.27%S to 0.89%S. The four suspensions (never-dried, free of added electrolyte) all underwent liquid crystalline phase separation, but the concentration onset for the emergence of the chiral nematic phase shifted to higher values with increasing surface charge. Similarly, suspension viscosity was also influenced by surface charge, with suspensions of lower surface charge CNCs more viscous and tending to gel at lower concentrations. The properties of the suspensions were interpreted in terms of the increase in effective diameter of the nanocrystals due to the surface electrostatic repulsion of the negative sulfate half-esters, as modified by the screening effects of the H+ counterions in the suspensions. The results suggest that there is a threshold surface charge density (∼0.3%S) above which effective volume considerations are dominant across the concentration range relevant to liquid crystalline phase formation. Above this threshold value, phase separation occurs at the same effective volume fraction of CNCs (∼10 vol %), with a corresponding increase in critical concentration due to the decrease in effective diameter that occurs with increasing surface charge. Below or near this threshold value, the formation of end-to-end aggregates may favor gelation and interfere with ordered phase formation.

DNA-Metalization: Synthesis and Properties of Novel Silver-Containing DNA Molecules (Adv. Mater. 24/2016).

D. Porath, A. Kotlyar, and co-workers transform DNA to a conducting material by metalization through coating or chemical modifications, as described on page 4839. Specific and reversible metalization of poly(dG)-poly(dC) DNA by migration of atoms from silver nanoparticles to the DNA is demonstrated. As the transformation occurs gradually, novel, truly hybrid molecular structures are obtained, paving the way to their usage as nanowires in programmable molecular electronic devices and circuits.

Controlling Anisotropic Growth of Colloidal ZnSe Nanostructures

Semiconductor nanocrystals serve as outstanding model systems for studying quantum confined size and shape effects. Shape control is an important knob for controlling their properties but so far it has been well developed mainly for heavy-metal containing semiconductor nanocrystals, limiting their further widespread utilization. Herein, we report a synthesis of heavy-metal free ZnSe nanocrystals with shape and size control through utilization of well-defined molecular clusters. In this approach, ZnSe nanowires are synthesized and their length and shape control is achieved by introduction of controlled amounts of molecular clusters. As a result of [Zn4(SPh)10](Me4N)2 clusters (Zn4 clusters) addition, short ZnSe nanorods or ZnSe nanodots can be obtained through tuning the ratio of Zn4 clusters to ZnSe. A study using transmission electron microscopy revealed the formation of a hybrid inorganic-organic nanowire, whereby the ligands form a template for self-assembly of ZnSe magic size clusters. The hybrid nanowire template becomes shorter and eventually disappears upon increasing amount of Zn4 clusters in the reaction. The generality of the method is demonstrated by using isostructural [Cu4(SPh)6](Me4N)2 clusters, which presented a new approach to Cu doped ZnSe nanocrystals and provided also a unique opportunity to employ X-ray absorption fine structure spectroscopy for deciphering the changes in the local atomic-scale environment of the clusters and explaining their role in the process of the nanorods formation. Overall, the introduction of molecular clusters presented here opens a path for growth of colloidal semiconductor nanorods, expanding the palette of materials selection with obvious implications for optoelectronic and biomedical applications.

Selective staining and eradication of cancer cells by protein-carrying DARPin-functionalized liposomes

The proteoliposomes containing large quantities of highly fluorescent protein, mCherry or a fragment of Pseudomonas exotoxin A, PE40 and functionalized with the designed ankyrin repeat protein (DARPin), which targets human epidermal growth factor receptor 2 (HER2) were shown to specifically stain and kill in sub‐nanomolar concentrations human breast adenocarcinoma cells, overexpressing HER2, respectively. ABSTRACT Since their discovery, liposomes have been widely employed in biomedical research. These nano‐size spherical vesicles consisting one or few phospholipid bilayers surrounding an aqueous core are capable of carrying a wide variety of bioactive compounds, including drugs, peptides, nucleic acids, proteins and others. Despite considerable success achieved in synthesis of liposome constructs containing bioactive compounds, preparation of ligand‐targeted liposomes comprising large quantities of encapsulated proteins that are capable of affecting pathological cells still remains a big challenge. Here we described a novel method for preparation of small (80–90nm in diameter) unilamellar liposomes containing very large quantities (thousands of protein molecules per liposome) of heme‐containing cytochrome c, highly fluorescent mCherry and highly toxic PE40 (Pseudomonas aeruginosa Exotoxin A domain). Efficient encapsulation of the proteins was achieved through electrostatic interaction between positively charged proteins (at pH lower than pI) and negatively charged liposome membrane. The proteoliposomes containing large quantities of mCherry or PE40 and functionalized with designed ankyrin repeat protein (DARPin)_9–29, which targets human epidermal growth factor receptor 2 (HER2) were shown to specifically stain and kill in sub‐nanomolar concentrations HER2‐positive cells, overexpressing HER2, respectively. Specific staining and eradication of the receptor‐positive cells demonstrated here makes the DARPin‐functionalized liposomes carrying large quantities of fluorescent and/or toxic proteins a promising candidate for tumor detection and therapy.

Structure, Assembly, and Disassembly of Tubulin Single Rings

Single and double tubulin rings were studied under a range of conditions and during microtubule (MT) assembly and disassembly. Here, tubulin was purified from porcine brain and used without any further modifications or additives that promote ring assembly. The structure of single GDP-rich tubulin rings was determined by cryo-transmission electron microscopy and synchrotron solution X-ray scattering. The scattering curves were fitted to atomic models, using our state-of-the-art analysis software, D+ . We found that there is a critical concentration for ring formation, which increased with GTP concentration with temperature. MT assembly or disassembly, induced by changes in temperature, was analyzed by time-resolved small-angle X-ray scattering. During MT assembly, the fraction of rings and unassembled dimers simultaneously decreased. During MT disassembly, the mass fraction of dimers increased. The increase in the concentration of rings was delayed until the fraction of dimers was sufficiently high. We verified that pure dimers, eluted via size-exclusion chromatography, could also form rings. Interestingly, X-ray radiation triggered tubulin ring disassembly. The concentration of disassembled rings versus exposure time followed a first-order kinetics. The disassembly rate constant and initial concentration were determined. X-ray radiation-triggered disassembly was used to determine the concentration of rings. We confirmed that following a temperature jump, the mass fraction of rings decreased and then stabilized at a constant value during the first stage of the MT assembly kinetics. This study sheds light on the most basic assembly and disassembly conditions for in vitro single GDP-rich tubulin rings and their relation to MT kinetics.