Single-Step Purification of Monomeric l-Selectin via Aptamer Affinity Chromatography

l-selectin is a transmembrane receptor expressed on the surface of white blood cells and responsible for the tethering of leukocytes to vascular endothelial cells. This initial intercellular contact is the first step of the complex leukocyte adhesion cascade that ultimately permits extravasation of leukocytes into the surrounding tissue in case of inflammation. Here we show the binding of a soluble histidine tagged l-selectin to a recently described shortened variant of an l-selectin specific DNA aptamer with surface plasmon resonance. The high specificity of this aptamer in combination with its high binding affinity of ~12 nM, allows for a single-step protein purification from cell culture supernatants. In comparison to the well-established Ni-NTA based technology, aptamer affinity chromatography (AAC) was easier to establish, resulted in a 3.6-fold higher protein yield, and increased protein purity. Moreover, due to target specificity, the DNA aptamer facilitated binding studies directly from cell culture supernatant, a helpful characteristic to quickly monitor successful expression of biological active l-selectin. View Full-Tex

Polymeric near-infrared absorbing dendritic nanogels for efficient in vivo photothermal cancer therapy

In recent years, several near-infrared light absorbing inorganic nanomaterials have been developed for photothermal therapy. However, their biological fate after injection limits their clinical utilization. In this work, we developed a novel polymeric near-infrared light absorbing material based on a biocompatible thermoresponsive nanogel that is semi-interpenetrated with polyaniline, a conjugated polymer with strong near-infrared absorbance. This polymeric nanocomposite generates heat after being irradiated by NIR light, thereby inducing a local hyperthermia that is used for photothermal cancer therapy in vitro and in vivo

Dendritic polymer imaging systems for the evaluation of conjugate uptake and cleavage

Fluorescent turn-on probes combined with polymers have a broad range of applications, e.g. for intracellular sensing of ions, small molecules, or DNA. In the field of polymer therapeutics, these probes can be applied to extend the in vitro characterization of novel conjugates beyond cytotoxicity and cellular uptake studies. This is particularly true in cases in which polymer conjugates contain drugs attached by cleavable linkers. Better information on the intracellular linker cleavage and drug release would allow a faster evaluation and optimization of novel polymer therapeutic concepts. We therefore developed a fluorescent turn-on probe that enables direct monitoring of pH-mediated cleavage processes over time. This is achieved by exploiting the fluorescence resonance energy transfer (FRET) between two dyes that have been coupled to a dendritic polymer. We demonstrate the use of this probe to evaluate polymer uptake and intracellular release of cargo in a cell based microplate assay that is suitable for high throughput screening

Matrix Metalloproteinase-sensitive Multistage Nanogels Promote Drug Transport in 3D Tumor Model

Physiological barriers inside of tumor tissue often result in poor interstitial penetration and heterogeneous intratumoral distribution of nanoparticle-based drug delivery systems (DDS). Novel, matrix metalloproteinase (MMP)-sensitive peptide-crosslinked nanogels (pNGs) as multistage DDS are reported with a beneficial size reduction property to promote the process of deep tissue penetration. Methods: The presented pNGs are based on a dendritic polyglycerol (dPG) scaffold crosslinked by a modified MMP-sensitive fluorogenic peptide. The crosslinker integrates degradability in response to proteases present in the tumor microenvironment. Surfactant-free, inverse nanoprecipitation is employed to prepare the nanogels using strain-promoted click chemistry. The size and crosslinking density of the pNGs are controlled by the functionalization degree of dPG with cyclooctyne groups and by the peptide crosslinker fraction. The intrinsic reporter moiety of the crosslinker was used to study the influence of pNG compositions on the degradation profile. The therapeutic drug Doxorubicin was conjugated through a pH-sensitive linkage to dPG to form a multistage DDS. The penetration behavior of the pNGs was studied using agarose matrix and multicellular tumor spheroids (MCTS). Results: Nanogel sizes were controlled in the range of 150-650 nm with narrow size distributions and varying degrees of crosslinking. The pNGs showed stability in PBS and cell media but were readily degraded in the presence of MMP-7. The crosslinking density influenced the degradation kinetic mediated by MMP-7 or cells. Stable conjugation of DOX at physiological pH and controlled drug release at acidic pH were observed. The digestions of nanogels lead to a size reduction to polymer-drug fragments which efficiently penetrated into agarose gels. Moreover, the degradable multistage pNGs demonstrated deeper penetration into MCTS as compared to their non-degradable counterparts. Thus, degradable pNGs were able to deliver their cargo and efficiently reduce the cell viability in MCTS. Conclusion: The triggered size reduction of the pNGs by enzymatic degradation can facilitate the infiltration of the nanocarrier into dense tissue, and thereby promote the delivery of its cargo

A fast open-source Fiji-macro to quantify virus infection and transfection on single-cell level by fluorescence microscopy

The ability to automatically analyze large quantities of image data is a valuable tool for many biochemical assays, as it rapidly provides reliable data. Here, we describe a fast and robust Fiji macro for the analysis of cellular fluorescence microscopy images with single-cell resolution. The macro presented here was validated by successful reconstruction of fluorescent and non-fluorescent cell mixing ratios (for fluorescence fractions ranging between 0 and 100%) and applied to quantify the efficiency of transfection and virus infection inhibition. It performed well compared with manually obtained image quantification data. Its use is not limited to the cases shown here but is applicable for most monolayered cellular assays with nuclei staining. We provide a detailed description of how the macro works and how it is applied to image data. It can be downloaded free of charge and may be used by and modified according to the needs of the user. • Rapid, simple, and reproducible segmentation of eukaryotic cells in confluent cellular assays • Open-source software for use without technical or computational expertise • Single-cell analysis allows identification and quantification of virus infected cell populations and infection inhibitio

Significantly enhanced proteolytic activity of cyclen complexes by monoalkylation

A simple approach towards efficient artificial proteases based on the cyclen ligand is presented. We thus achieved an increase of the proteolytic activity of two orders of magnitude when compared to the unsubstituted cyclen complex. Amphiphilic Cu(II) and Co(III) complexes cut BSA and myoglobin as model substrates at μM concentrations. MALDI-ToF MS is used to identify the cleavage fragments

Forty Years after the Discovery of Its Nucleolytic Activity: [Cu(phen)(2)](2+) Shows Unattended DNA Cleavage Activity upon Fluorination

[Cu(phen)(2)](2+) (phen=1,10-phenanthroline) is the first and still one of the most efficient artificial nucleases. In general, when the phen ligand is modified, the nucleolytic activity of its Cu-II complex is significantly reduced. This is most likely due to higher steric bulk of such ligands and thus lower affinity to DNA. Cu-II complexes with phen ligands having fluorinated substituents (F, CF3, SF5, SCF3) surprisingly showed excellent DNA cleavage activity-in contrast to the unsubstituted [Cu(phen)(2)](2+)-in the absence of the otherwise required classical, bioabundant external reducing agents like thiols or ascorbate. This nucleolytic activity correlates well with the half-wave potentials E-1/2 of the complexes. Cancer cell studies show cytotoxic effects of all complexes with fluorinated ligands in the low mu m range, whereas they were less toxic towards healthy cells (fibroblasts)

Polyglutamic acid-based Crosslinked Doxorubicin Nanogels as an Anti-Metastatic Treatment for Triple Negative Breast Cancer

PreprintTreatment of triple negative breast cancer (TNBC)-associated metastasis represents an unmet clinical need, and we lack effective therapeutics for a disease that exhibits high relapse rates and associates with poor patient outcomes. Advanced nanosized drug delivery systems may enhance the efficacy of first-line chemotherapeutics by altering drug pharmacokinetics and enhancing tumor/metastasis targeting to signif-icantly improve efficacy and safety. Herein, we propose the application of injectable poly-amino acid-based nanogels (NGs) as a versatile hydrophilic drug delivery platform for the treatment of TNBC lung metastasis. We prepared biocompatible and biodegradable cross-linked NGs from polyglutamic acid (PGA) loaded with the chemotherapeutic agent doxorubicin (DOX). Our optimized synthetic procedures generated NGs of ~100 nm in size and 25 wt% drug loading content that became rapidly internalized in TNBC cell lines and displayed IC50 values comparable to the free form of DOX. Importantly, PGA-DOX NGs significantly inhibited lung metastases and almost completely suppressed lymph node metastases in a spontaneously metastatic orthotopic mouse TNBC model. Overall, our newly developed PGA-DOX NGs represent a potentially effective therapeutic strategy for the treatment of TNBC metastases.A.S-H thanks MC IEF actions (Project 302717). We thank Dr. M. A. Molina for AFM experiments, Ser-vicio SEM Cordoba (Argentina), and Mario Soriano Navarro from the electron microscopy service for Cryo-TEM pictures at CIPF. The authors would also like to thank Dr. Stuart P. Atkinson for his collabo-ration in the revision of the manuscript. This work has been supported by the European Research Council (grant ERC-CoG-2014-648831 “MyNano”), by the Spanish Ministry of Science and Innovation (SAF2013-44848-R, SAF2016-80427-R, RTI2018-099227-B-I00), by a Marie Curie IEF (Project 302717), and the Bundesministerium für Bildung und Forschung (BMBF) through the NanoMatFutur award (13N12561). Part of the equipment employed in this work has been funded by Generalitat Valen-ciana and co-financed with FEDER funds (PO FEDER of Comunitat Valenciana 2014–2020

Exploiting cyanine dye J-aggregates/monomer equilibrium in hydrophobic protein pockets for efficient multi-step phototherapy: An innovative concept for smart nanotheranostics

After several decades of development in the field of near-infrared (NIR) dyes for photothermal therapy (PTT), indocyanine green (ICG) still remains the only FDA-approved NIR contrast agent. However, upon NIR light irradiation ICG can react with molecular oxygen to form reactive oxygen species and degrade the ICG core, losing the convenient dye properties. In this work, we introduce a new approach for expanding the application of ICG in nanotheranostics, which relies on the confinement of self-organized J-type aggregates in hydrophobic protein domains acting as monomer depots. Upon the fast photobleaching, while the dye is irradiated, this strategy permits the equilibrium-driven monomer replacement after each irradiation cycle that radically increases the systems' effectivity and applicability. Gadolinium-doped casein micelles were designed to prove this novel concept at the same time as endowing the nanosystems with further magnetic resonance imaging (MRI) ability for dual-modal imaging-guided PTT. By teaching a new trick to a very old dog, the clinical prospect of ICG will undoubtedly be boosted laying the foundation for novel therapeutics. It is anticipated that future research could be expanded to other relevant J-aggregates-forming cyanine dyes or nanocrystal formulations of poorly water-soluble photosensitizers.Fil: Picchio, Matías Luis. Universidad Nacional de Córdoba. Instituto de Investigación y Desarrollo en Ingeniería de Procesos y Química Aplicada. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación y Desarrollo en Ingeniería de Procesos y Química Aplicada; ArgentinaFil: Bergueiro, Julian. Freie Universität Berlin; AlemaniaFil: Wedepohl, Stefanie. Freie Universität Berlin; AlemaniaFil: Minari, Roque Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; ArgentinaFil: Alvarez Igarzabal, Cecilia Ines. Universidad Nacional de Córdoba. Instituto de Investigación y Desarrollo en Ingeniería de Procesos y Química Aplicada. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación y Desarrollo en Ingeniería de Procesos y Química Aplicada; ArgentinaFil: Gugliotta, Luis Marcelino. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; ArgentinaFil: Cuggino, Julio César. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; ArgentinaFil: Calderón, Marcelo. Polymat; Españ

Crosslinked casein-based micelles as a dually responsive drug delivery system

New types of biodegradable nanocarriers for drug delivery were prepared using casein (CAS) micelles as particle templates and glyceraldehyde (GAL) as a crosslinking agent. We found that highly crosslinked casein micelles (CCM) maintained their structural integrity at pH 7.4 (plasma conditions) but were easily degraded in the presence of proteases at pH 5 (lysosomal conditions). Nile red (NR) was chosen as a hydrophobic model drug inspired by the natural role of casein as lipophilic nutrient nanotransporter. The cumulative release of the NR-loaded micelles showed marginal dye leakage at pH 7.4 but was significantly accelerated by protease and pH-mediated degradation of the nanocarriers in a dual-responsive fashion. The prepared nanocarriers possess many favorable features for drug delivery: excellent biocompatibility and biodegradability, high stability in physiological conditions, remarkable capacity for the encapsulation of hydrophobic drugs, minimal drug leakage under extracellular conditions, and rapid drug release in response to the endo-lysosomal levels of pH and proteases. In this regard, the prepared CCM represent a promising candidate for the delivery and triggered release of anti-cancer drugs in lysosomal environments.Fil: Picchio, Matías Luis. Universidad Nacional de Córdoba. Instituto de Investigación y Desarrollo en Ingeniería de Procesos y Química Aplicada. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación y Desarrollo en Ingeniería de Procesos y Química Aplicada; ArgentinaFil: Cuggino, Julio César. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; ArgentinaFil: Nagel, Gregor. Freie Universitat Berlin; AlemaniaFil: Wedepohl, Stefanie. Freie Universitat Berlin; AlemaniaFil: Minari, Roque Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; ArgentinaFil: Alvarez Igarzabal, Cecilia Ines. Universidad Nacional de Córdoba. Instituto de Investigación y Desarrollo en Ingeniería de Procesos y Química Aplicada. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación y Desarrollo en Ingeniería de Procesos y Química Aplicada; Argentina. Freie Universitat Berlin; AlemaniaFil: Gugliotta, Luis Marcelino. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; ArgentinaFil: Calderon, Marcelo. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Química Orgánica; Argentina. Freie Universitat Berlin; Alemani