Ofra Benny

A. Fluksman, E. Steinberg, N. Orehov, E. Shai, A. Lahiani, J. Katzhendler, C. Marcinkiewicz, P. Lazarovici, and O. Benny. 2020. “Integrin α2β1-Targeted Self-Assembled Nanocarriers for Tumor Bioimaging.” ACS Applied Bio Materials, 3, 9, Pp. 6059-6070. Publisher's Version Abstract
Recent developments in near-infrared (NIR) dyes and imaging modalities enable tumor fluorescent images in preclinical and clinical settings. However, NIR dyes have several drawbacks, and therefore, there is an unmet diagnostic need for NIR dye encapsulation in appropriate pharmaceutical nanocarriers with targeting abilities for the purpose of achieving effective diagnosis and image-guided surgeries. Because integrin receptors are established diagnostic targets, the cyclic Arg-Gly-Asp (RGD) peptides, recognizing the αVβ3 integrin, have been extensively investigated for radiology and bioimaging of tumors. However, the Lys(Arg)-Thr-Ser [K(R)TS] cyclic peptides, selective for collagen receptors α1β1/α2β1 integrins, which are overexpressed in many tumors, were not yet investigated and therefore used here for tumor bioimaging with a unique α2β1-integrin-targeted nanocarrier, encapsulating the indocyanine green NIR dye. We synthesized three kinds of peptides: two cyclic RTS peptides functional only in the cyclic conformation and a linear peptide lacking the cyclic cysteine constrained RTS loop. We used them for the preparation of integrin-targeted self-assembled nanocarriers (ITNCs), referred to as OF5 and OF27, and a nontargeted control nanocarrier, referred to as OF70. Their selective association was demonstrated with α2β1 integrin expressing cell cultures and three-dimensional tumor spheroids and by competition with a α2β1 selective disintegrin. Cytotoxicity experiments in vitro demonstrated the safety of the ITNCs. The targeting potential and the biodistribution of the ITNCs, applied intravenously in A431 tumor-bearing nude mice, were evaluated in vivo using NIR bioimaging. Time-dependent biodistributions indicated that the ITNC OF27 showed higher fluorescent signals in main tissues, with no cytotoxic effects to major organs, and presented higher accumulation in tumors. Cumulatively, these results highlight the potential of the ITNC OF27 as an optical and innovative pharmaceutical bioimaging system, suitable for integrin α2β1 receptor in vivo tumor targeting and visualization in the NIR region. © 2020 American Chemical Society.
The critical micelle concentration (CMC) is the point at which micelles are self-assembled from surfactants added to the system. Determination of the CMC is commonly based on surface tension and conductivity measurements since these are relatively simple and well described. The dye micellization method performed using pyrene as a fluorescent probe, is limited like other methods by inaccuracy and the lack of a standard procedure. We established a simple, robust, and straightforward method based on dye micellization, using coumarin-6 as a fluorescent probe for CMC determination. We first optimized the method and demonstrated the problematic pyrene detection by a plate reader. We compared the novel method to the established surface tension method using anionic, cationic and non-ionic surfactants, and compared to reported values in the literature. We selected to measure sodium dodecyl sulfate using the conductivity method, followed by visualization by fluorescence microscopy. The values obtained by our method were reproducible and in accordance with the values reported in the literature, regardless of the ionic charge of the surfactant. Adopting such a robust protocol would be beneficial for pharmaceutical research and industry and has the potential to replace the more laborious and less accurate commonly available methods. © 2019 The Royal Society of Chemistry.
Polymeric porous particles are currently used for various applications in biotechnology, tissue engineering and pharmaceutical science, e.g., floating drug delivery systems and inhaled formulations. Particle shape and size depend on variable parameters; among them, polymer type and concentration, stirring speed, pH and type of solvent. In this study, porous poly(lactic-co-glycolic) acid (PLGA) and poly(D,L-lactide) (PLA) microspheres (MPs), with varying sizes and morphologies, were synthesized and optimized using both batch formulation and a flow-focusing microfluidic device. A well-established method of preparation utilizing solvent evaporation and the double emulsion technique was performed. Similar to other batch encapsulation methods, this technique is time and reagent consuming and consists of several steps. Hence, although porous structures provide tremendous opportunity in the design of new applications for tissue engineering and as improved controlled-release carriers, the synthesis of these particles with predefined properties remains challenging. We demonstrated the fabrication of porous MPs using a simple microfluidic device, compared to batch synthesis fabrication; and the effect of solvent, polymer concentration and type, post-hydrolysis treatment, on porosity degree. Moreover, a kinetic release study of fluorescent molecule was conducted for non-porous in comparison to porous particles. An overview of future prospects and the potential of these porous beads in this scientific area are discussed. © 2019 by the authors.