17 research outputs found
Multivalent interaction and selectivities in selectin binding of functionalized gold colloids decorated with carbohydrate mimetics
Colloidal gold particles with functionalized organic shells were applied as novel selectin binders. The ligand shell was terminated with different monocyclic carbohydrate mimetics as simplified analogs of the sLe(x) unit found in biological selectin ligands. The multivalent presentation of the sulfated selectin binding epitopes on the gold particles led to extremely high binding affinities towards L- and P-selectin and IC(50) values in the subnanomolar range. Depending on the ring size of the sulfated carbohydrate mimetic, its substitution pattern and its configuration, different selectivities for either L-selectin or P-selectin were obtained. These selectivities were not found for gold particles with simple acyclic sulfated alcohols, diols and triols in the ligand shell. In addition, the influence of the particle size and the thickness of the hydrophobic organic shell were systematically investigated
Controlling genetic heterogeneity in gene-edited hematopoietic stem cells by single-cell expansion
Gene editing using engineered nucleases frequently produces unintended genetic lesions in hematopoietic stem cells (HSCs). Gene-edited HSC cultures thus contain heterogeneous populations, the majority of which either do not carry the desired edit or harbor unwanted mutations. In consequence, transplanting edited HSCs carries the risks of suboptimal efficiency and of unwanted mutations in the graft. Here, we present an approach for expanding gene-edited HSCs at clonal density, allowing for genetic profiling of individual clones before transplantation. We achieved this by developing a defined, polymer-based expansion system and identifying long-term expanding clones within the CD201 +CD150 +CD48 -c-Kit +Sca-1 +Lin - population of precultured HSCs. Using the Prkdc scid immunodeficiency model, we demonstrate that we can expand and profile edited HSC clones to check for desired and unintended modifications, including large deletions. Transplantation of Prkdc-corrected HSCs rescued the immunodeficient phenotype. Our ex vivo manipulation platform establishes a paradigm to control genetic heterogeneity in HSC gene editing and therapy
Cancer-selective, single agent chemoradiosensitising gold nanoparticles
Two nanometre gold nanoparticles (AuNPs), bearing sugar moieties and/or thiol-polyethylene glycol-amine (PEG-amine), were synthesised and evaluated for their in vitro toxicity and ability to radiosensitise cells with 220 kV and 6 MV X-rays, using four cell lines representing normal and cancerous skin and breast tissues. Acute 3 h exposure of cells to AuNPs, bearing PEG-amine only or a 50:50 ratio of alpha-galactose derivative and PEG-amine resulted in selective uptake and toxicity towards cancer cells at unprecedentedly low nanomolar concentrations. Chemotoxicity was prevented by co-administration of N-acetyl cysteine antioxidant, or partially prevented by the caspase inhibitor Z-VAD-FMK. In addition to their intrinsic cancer-selective chemotoxicity, these AuNPs acted as radiosensitisers in combination with 220 kV or 6 MV X-rays. The ability of AuNPs bearing simple ligands to act as cancer-selective chemoradiosensitisers at low concentrations is a novel discovery that holds great promise in developing low-cost cancer nanotherapeutics
Multivalent interaction and selectivities in selectin binding of functionalized gold colloids decorated with carbohydrate mimetics
Surface functionalization of nanoparticles for the directed interaction with biomolecules
Die Nanotechnologie hat in den letzten Jahren deutlich an Interesse gewonnen.
Obwohl sie noch am Anfang ihrer Entwicklung steht, zeigen erste erfolgreiche
Anwendungen [13] ihr großes Potenzial. Das Interesse an Nanomaterialien in den
verschiedensten Bereichen beruht auf den einzigartigen optischen,
elektronischen und katalytischen Eigenschaften, welche durch eine
Nanostrukturierung erzielt werden können [102]. Das große Oberfläche-zu-
Volumen-Verhältnis macht Nanomaterialien auch zu idealen Templaten für die
Präsentation funktioneller Gruppen. In der vorliegenden Arbeit wurden NP
unterschiedlicher Größe mit schmalen Größenverteilungen für zwei
unterschiedliche Anwendungsbereiche synthetisiert und funktionalisiert.
Beruhend auf elektrostatischen Wechselwirkungen wurde im ersten Teil der
vorliegenden Arbeit eine Peptid-induzierte Aggregation von NP vorgestellt. Die
Aggregation wurde dabei durch Zugabe verschiedener Peptide, welche eine
coiled-coil-Struktur aufwiesen, induziert, die Struktur der sich bildenden
Aggregate mit verschiedenen Methoden untersucht und durch Variation
verschiedener Parameter die Möglichkeiten zum Aufbau geordneter Architekturen
aus diesen Aggregaten analysiert. Die Verwendung von Peptiden als
strukturinduzierendem Baustein zur Erzeugung schaltbarer mehrdimensionaler NP-
Architekturen hat sich dabei als erfolgreich erwiesen. Die Schaltbarkeit
dieses Materials wurde durch eine pH-Abhängigkeit der coiled-coil-Struktur des
Peptids erzielt. Mittels pH-Titration konnte außerdem gezeigt werden, dass es
sich bei dem Bildungs- und Auflösungsprozess der NP-Aggregate um einen
reversiblen Prozess handelt. Die Notwendigkeit der coiled-coil Struktur des
Peptids für die Ausbildung der Aggregate wurde durch Versuche mit einem
ungefalteten Peptid gleicher Aminosäurenzusammensetzung, aber veränderter
Sequenz, nachgewiesen. Im zweiten Teil der vorliegenden Arbeit wurde die
Verwendung unterschiedlich funktionalisierter Au-NP als Selektin-Inhibitoren
vorgestellt. Die multivalente Präsentation Selektin-bindender Strukturen auf
den NP führte zu einer deutlichen Verbesserung der Bindungseigenschaften im
Vergleich zu den entsprechenden monovalenten Molekülen. Durch Trägerung auf
den NP konnte eine Bindungsverstärkung um den Faktor 106 erzielt werden.
Selbst die Trägerung nicht bindender Substanzen führte zu NP mit starken
inhibitorischen Eigenschaften. Eine Sulfatierung der NP wirkte sich nicht nur
positiv auf die Löslichkeit der NP in wässrigem Medium, sondern auch auf die
Bindungseigenschaften an L- und P-Selektin aus. Mit geeignet
funktionalisierten Au-NP wurden IC50-Werte für L- und P-Selektin im
pikomolaren Bereich erzielt. Als entscheidend für eine starke Selektin-NP-
Bindung hat sich dabei nicht nur eine polyanionische Struktur der
Ligandenhülle, sondern auch die Ausrichtung der negativen Ladungen auf der NP-
Oberfläche erwiesen. Obwohl auch mit einfachen Strukturen wie beispielsweise
sulfatiertem Serinol beeindruckende IC50-Werte erzielt wurden, bleibt die
Immobilisierung komplexerer Strukturen wie Kohlenhydratmimetika sehr
interessant, denn mit diesen NP ließ sich eine Selektivität des
Bindungsverhaltens zwischen L- und P-Selektin realisieren. Die
funktionalisierten NP wiesen keine Zelltoxizität auf und sind somit für die
Anwendung in vivo geeignet. In weiteren Tests muss nun das Verhalten der
Partikel in vivo untersucht werden.The interest in nanotechnology has significantly increased in recent years.
Although still at the beginning of its development first successful results
[13] indicate the big potential of this research area. The interest in
nanomaterials results from the unique optical, electronic and catalytic
properties, which can be achieved by nanostructuring [102]. Due to their
remarkably big surface-to-volume ratio nanomaterials are well suited as
templates for the presentation of functional groups. In this work
nanoparticles of different sizes with small size distributions have been
synthesized and functionalized for two types of applications. In the first
part of this work the peptide-induced aggregation of nanoparticles based on
electrostatic interactions was presented. The aggregation was induced by
addition of different coiled-coil-structured peptides. The structure of the
forming aggregates was investigated by different methods. The possibilities
for building regular structures of this material were analyzed by variation of
several parameters. The use of peptides as structure-imposing building blocks
for the creation of switchable multidimensional nanoparticle architectures was
successful. The switchability of this material was achieved by the pH-
dependency of the peptides coiled-coil-structure. Furthermore, the
reversibility of the formation and disaggregation process was shown by pH-
titration. The necessity of the peptide’s coiled-coil-structure for the
formation of aggregates was demonstrated by experiments with an unfolded
peptide composed of the same amino acids but in different sequence. In the
second part of this work the application of different functionalized gold
nanoparticles for the inhibition of selectins was presented. The multivalent
presentation of selectin-binding structures on the surface of nanoparticles
led to highly improved binding properties compared to the monovalent
structures. An amplification of binding by a factor of 106 was achieved by
immobilization on gold nanoparticles. Even the immobilization of non-binding
structures led to particles with strong inhibitory effects. Sulphation of the
particles improved not only the solubility in aqueous media, but also the
binding properties to L- and P-selectin. IC50 values in the picomolar range
were thus achieved. The polyanionic structure of the ligand shell in
combination with the arrangement of the negative charges on the particle
surface were decisive for a strong selectin-nanoparticle-binding. Although as
simple structures as sulphated serinol also yielded impressive IC50 values the
immobilization of more complex structures like carbohydrate mimetics remains
interesting because with these structures a selectivity of the binding
properties to L- and P-selectin can be realized. The functionalized particles
showed no cell toxicity and thereby proved their applicability in vivo.
Further tests are necessary to investigate the behaviour of the particles in
vivo
Clonogenic assay dose-response of adherent MCF-7 and MCF-10 cells exposed for 3 h to different concentrations of 50:50 or 0:100 αGal:PEG-amine AuNPs.
<p>The graphs represent the percentage of cell colonies compared to the no-nanoparticle control ±SEM.</p
TEM images of A,B) HSC-3 and C,D) HaCaT cells incubated for 3 h with 10 μg/ml 0:100 αGal:PEG-amine AuNPs.
<p>Boxed area in A is shown magnified in B. Scale bars A,C are 500 nm; B,D are 100 nm.</p
Hydroxyl radical formation assay of three different 6 μg/ml AuNP preparations in water with or without exposure to 10 Gy of A) 6 MV X-rays or B) 220 keV X-rays.
<p>Significant differences in fluorescence of 7-OHCCA probe following irradiation, compared to irradiated water only are indicated as <i>P</i><0.05 *, <i>P</i><0.01 **, <i>P</i><0.001 *** (ANOVA with Dunnett’s multiple comparisons post test).</p
Clonogenic assay of (A,B) MCF-7 and (C,D) MCF-10 cells following exposure to αGal:PEG-amine AuNPs at their MCF-7 IC50 concentrations, then different doses of A,C) 220 kV X-rays or B,D) 6 MV X-rays.
<p>Clonogenic assay of (A,B) MCF-7 and (C,D) MCF-10 cells following exposure to αGal:PEG-amine AuNPs at their MCF-7 IC50 concentrations, then different doses of A,C) 220 kV X-rays or B,D) 6 MV X-rays.</p