INMdok (Leibniz Institute for New Materials)
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Water-Driven Sol-Gel Transition in Native Cellulose/1-Ethyl-3-methylimidazolium Acetate Solutions
Full text linkThe addition of water to native cellulose/1-ethyl-3-methylimidazolium acetate solutions catalyzes the formation of gels, where polymer chain–chain intermolecular associations act as cross-links. However, the relationship between water content (Wc), polymer concentration (Cp), and gel strength is still missing. This study provides the fundamentals to design water-induced gels. First, the sol–gel transition occurs exclusively in entangled solutions, while in unentangled ones, intramolecular associations hamper interchain cross-linking, preventing the gel formation. In entangled systems, the addition of water has a dual impact: at low water concentrations, the gel modulus is water-independent and controlled by entanglements. As water increases, more cross-links per chain than entanglements emerge, causing the modulus of the gel to scale as Gp ∼ Cp2Wc3.0±0.2. Immersing the solutions in water yields hydrogels with noncrystalline, aggregate-rich structures. Such water–ionic liquid exchange is examined via Raman, FTIR, and WAXS. Our findings provide avenues for designing biogels with desired rheological properties
Evaluation of the Transport and Binding of Dopamine-Loaded PLGA Nanoparticles for the Treatment of Parkinson’s Disease Using In Vitro Model Systems
Full text linkThe treatment of Parkinson’s disease has been moving into the focus of pharmaceutical development. Yet, the necessity for reliable model systems in the development phase has made research challenging and in vivo models necessary. We have established reliable, reproducible in vitro model systems to evaluate the binding and transport of dopamine-loaded PLGA nanoparticles for the treatment of Parkinson’s disease and put the results in context with comparable in vivo results. The in vitro models have provided similar results concerning the usability of the investigated nanoparticles as the previously used in vivo models and thus provide a good alternative in line with the 3R principles in pharmaceutical research
On the generation of force required for actin-based motility
Full text linkThe fundamental question of how forces are generated in a motile cell, a lamellipodium, and a comet tail is the subject of this note. It is now well established that cellular motility results from the polymerization of actin, the most abundant protein in eukaryotic cells, into an interconnected set of filaments. We portray this process in a continuum mechanics framework, claiming that polymerization promotes a mechanical swelling in a narrow zone around the nucleation loci, which ultimately results in cellular or bacterial motility. To this aim, a new paradigm in continuum multi-physics has been designed, departing from the well-known theory of Larché–Cahn chemo-transport-mechanics. In this note, we set up the theory of network growth and compare the outcomes of numerical simulations with experimental evidence
A New Class of Polyion Complex Vesicles (PIC-somes) to Improve Antimicrobial Activity of Tobramycin in Pseudomonas Aeruginosa Biofilms
Full text linkPseudomonas aeruginosa (PA) is a major healthcare concern due to its tolerance to antibiotics when enclosed in biofilms. Tobramycin (Tob), an effective cationic aminoglycoside antibiotic against planktonic PA, loses potency within PA biofilms due to hindered diffusion caused by interactions with anionic biofilm components. Loading Tob into nano-carriers can enhance its biofilm efficacy by shielding its charge. Polyion complex vesicles (PIC-somes) are promising nano-carriers for charged drugs, allowing higher drug loadings than liposomes and polymersomes. In this study, a new class of nano-sized PIC-somes, formed by Tob-diblock copolymer complexation is presented. This approach replaces conventional linear PEG with brush-like poly[ethylene glycol (methyl ether methacrylate)] (PEGMA) in the shell-forming block, distinguishing it from past methods. Tob paired with a block copolymer containing hydrophilic PEGMA induces micelle formation (PIC-micelles), while incorporating hydrophobic pyridyldisulfide ethyl methacrylate (PDSMA) monomer into PEGMA chains reduces shell hydrophilicity, leads to the formation of vesicles (PIC-somes). PDSMA unit incorporation enables unprecedented dynamic disulfide bond-based shell cross-linking, significantly enhancing stability under saline conditions. Neither PIC-somes nor PIC-micelles show any relevant cytotoxicity on A549, Calu-3, and dTHP-1 cells. Tob's antimicrobial efficacy against planktonic PA remains unaffected after encapsulation into PIC-somes and PIC-micelles, but its potency within PA biofilms significantly increases
Cation selectivity during flow electrode capacitive deionization
Full text linkEfficient separation of specific ions from aqueous media is crucial for advanced water treatment and resource recovery. Flow electrode capacitive deionization (FCDI) offers potential for selective ion removal through continuous operation. This study evaluates the performance of selective cation separation using a commercial activated carbon slurry in a multi-ion solution of monovalent (Li+, Na+, K+) and bivalent (Ca2+, Mg2+) cations. We assess ion removal and cation selectivity under different operational parameters, such as applied potential, slurry flow rate, and feed water flow rate. Our data show that bivalent cations, namely Ca2+ and Mg2+, are preferentially removal due to their higher charge-to-size ratio, aligning with hydrated ion sizes. The highest separation rate was observed for Ca2+ (5.7 μg cm−2 min−1), and the lowest for Li+ (0.2 μg cm−2 min−1). At the highest applied voltage (1.2 V), charge efficiencies reached 70 %, with an energy consumption of 41 Wh mol−1 for nearly complete cation removal. Optimal conditions were identified with a slurry flow rate of 6 mL min−1, feed water flow rate of 2 mL min−1, activated carbon content of 10 mass%, 1 mass% carbon black, and a cell voltage of 1.2 V. These findings highlight the importance of optimizing operational parameters to enhance ion removal
Thermo-amplifier circuit in probiotic E. coli for stringently temperature-controlled release of a novel antibiotic
Full text linkPeptide drugs have seen rapid advancement in biopharmaceutical development, with over 80 candidates approved globally. Despite their therapeutic potential, the clinical translation of peptide drugs is hampered by challenges in production yields and stability. Engineered bacterial therapeutics is a unique approach being explored to overcome these issues by using bacteria to produce and deliver therapeutic compounds at the body site of use. A key advan‑ tage of this technology is the possibility to control drug delivery within the body in real time using genetic switches. However, the performance of such genetic switches suffers when used to control drugs that require post‑translational modifications or are toxic to the host. In this study, these challenges were experienced when attempting to establish a thermal switch for the production of a ribosomally synthesized and post‑translationally modified peptide antibiotic, darobactin, in probiotic E. coli. These challenges were overcome by developing a thermo‑amplifier circuit that combined the thermal switch with a T7 RNA Polymerase. Due to the orthogonality of the Polymerase, this strategy overcame limitations imposed by the host transcriptional machinery. This circuit enabled production of pathogen‑inhibitory levels of darobactin at 40 °C while maintaining leakiness below the detection limit at 37 °C. Furthermore, the thermo‑amplifier circuit sustained gene expression beyond the thermal induction duration such that with only 2 h of induction, the bacteria were able to produce pathogen‑inhibitory levels of darobactin. This performance was maintained even in physiologically relevant simulated conditions of the intestines that include bile salts and low nutrient levels
Tetrazole Methylsulfone - Thiol crosslinked hydrogels for automated and high-throughput 3D cell culture
Full text linkThe understanding of diseases and the development of personalized therapies relies on the predictive potential of 3D cell culture models. Automation of the culture steps is crucial, for which in situ crosslinked hydrogels with adequate and tunable kinetics under physiological conditions are needed to temporally replace the natural 3D matrix. This Thesis investigates hydrogels formed by tetrazol methylsulfone (TzMS) derivatized star-polyethylene glycol and thiol-crosslinkers for automated 3D cell encapsulation, including precursor synthesis scale-up, characterization of their crosslinking kinetics and mechanical properties, and their semi-automated preparation with pipetting robots. The optimization of reaction and purification conditions allowed for an upscaling to 0.6-g scale of polymer precursor. The gelation kinetics and mechanical properties of the hydrogels were studied as a function of precursors stoichiometry, crosslinker architecture, biofunctionalization degree and pH. Tunability of gelation time and stiffness at pH 7.0 – 8.0 was explored for a cell-compatible hydrogel composition including the cell-adhesive ligand RGD and the enzyme-cleavable peptide VPM as crosslinker. A semi-automated pipetting protocol was set up to prepare 5 µl hydrogels in a 384-well plate format, and statistical experimental design was used to systematically minimize variability. The results demonstrate the suitability and limitations of TzMS/thiol chemistry for in situ cell encapsulation.Das Verständnis von Krankheiten und die Entwicklung personalisierter Therapien hängen von dem Fortschritt bei 3D-Zellkulturmodellen ab, bei denen in situ gebildete Hydrogele verwendet werden, um Zellen einzukapseln und die natürliche 3D-Matrix vorübergehend zu ersetzen. Von Vorteil sind Hydrogele mit kontrollierter Vernetzungskinetik, die in automatisierte Arbeitsabläufe implementiert werden können. Diese Dissertation befasst sich mit Hydrogelen, die aus Tetrazolmethylsulfon (TzMS)-derivatisiertem Stern-Polyethylenglykol mit kommerziellen thiolierten Vernetzern gebildet werden. Die bestehende fünfstufige Synthese und Aufreinigung der Hydrogel-Vorläufers wurde optimiert und hochskaliert. Die Vernetzungskinetik und mechanische Eigenschaften der Hydrogele wurden in Abhängigkeit von Stöchiometrie der Ausgangsstoffe, Vernetzerarchitektur, Biofunktionalisierungsgrad und pH-Wert untersucht. Die Einstellung von Vernetzungszeit und Steifigkeit wurden bei einem pH-Wert von 7,0 bis 8,0 für ein zellkompatibles Hydrogel mit dem zelladhäsiven Liganden RGD und dem enzymspaltbaren Peptid VPM als Vernetzer erforscht. Es wurde ein halbautomatisches Pipettierprotokoll für die Herstellung von 5 μl-Hydrogelen in einem 384-Well-Plattenformat erstellt, und die Variabilität mithilfe eines statistischer Versuchsplan systematisch minimiert. Die Ergebnisse zeigen die Eignung und die Grenzen der Thiol-TzMS-Chemie für die in situ Zellverkapselung
Polyacrylamide Hydrogels as Versatile Biomimetic Platforms to Study Cell-Materials Interactions
Full text linkPolyacrylamide (PAAm) hydrogels are widely adopted as 2D-model soft substrates for investigating cell-material interactions in a controlled in vitro environment. They offer facile synthesis, tunable physico-chemical properties, diverse biofunctionalization routes, optical transparency, mouldability in a range of geometries and shapes, and compatibility with living cells. PAAm hydrogels can be engineered to reconstruct physiologically relevant biointerfaces, like cell-matrix or cell–cell interfaces, featuring biochemical, mechanical, and topographical cues present in the extracellular environment. This Review provides a materials science perspective on PAAm material properties, fabrication, and modification strategies relevant to cell studies, highlighting their versatility and potential to address a wide range of biological questions. Current routes are presented to integrate cell-instructive features, such as 2D patterns, 2.5D surface topographies, or mechanical stiffness gradients. Finally, the recent advances are emphasized toward dynamic PAAm hydrogels with on-demand control over hydrogel properties as well as electrically conductive PAAm hydrogels for bioelectronics
Nanoparticle's shape is the game-Changer for a customized delivery through tunneling nanotubes among glioblastoma cells
No full textOver the past decade, increasing evidence suggested that cells are capable of establishing long distance communication routes with different function defined as Tunneling Nanotubes (TNTs). TNTs are thin, dynamic, long membrane protrusions that allow the intercellular exchanges of signal clues, molecules, organelles and pathogens. The presence of TNTs has been observed in several types of cancer, glioblastoma (GBM) included, where they emerge to steer a more malignant phenotype [1]. GBM is the most common malignant tumour of Central Nervous System (CNS), representing about 82% of cases of all malignant gliomas [2]. An innovative strategy that could represent a potential therapeutic approach is the targeting of tumour cells communication. Therefore, we are studying TNTs in GBM, to deepen both their structural and genesis features in order to exploit them to improve the intercellular distribution of nanomedicines in close and far away cells, thus reaching isolated tumour niches that are hardly targeted by simple drug diffusion in the brain parenchyma. Until now, different types of nanoparticles have been identified within TNTs. Very little is known about the role of fundamental physical parameters of nanoparticles such as size, charge, shape in determining their penetration across the BBB and their transfer between cells by TNTs. Considering that, TNTs thickness is in the range of 0.2-1 μm, it can be speculated that the size should not be a critical parameter while positively charged NPs could trigger the formation of TNTs due to a higher toxicity compared to those that are negatively charged. At the best of our knowledge, no data are available about the effect of NPs shape on the transfer efficiency between TNTs. For this purpose, spherical, discoidal and deformable nanoparticles were synthetized in order to evaluate if the nanoparticles shape could influence their ability to be transferred via TNTs. These nanoparticles were evaluated in 2D and 3D in vitro models composed of human GBM cells, carrying the EGFRvIII mutation and resistant to temozolomide [3]. The results showed that a single GBM cell is able to form more than one TNT and that TNTs are dynamic and transient structures. They can be actin or actin and α-tubulin positive, they can have a length between 20-100 μm with a thickness of 200-600 nm. Moreover, GBM TNTs are efficient in allowing the intercellular transport of the three different types of nanoparticles tested, in a bidirectional vesicles-free way. Nanoparticles were followed inside TNTs and their average and maximum velocity was evaluated. Moreover, through a co-culture assay it has been demonstrated that the shape affects the efficiency of the nanoparticles exchange via TNTs because the discoidal ones were those transferred most efficiently, in comparison to the other two nanoparticles. Additionally, we address the presence of the TNTs in 3D-tumour organoids. GBM cells grown in a 3D scaffold better recapitulate the features of patient-derived cells, in comparison to 2D culture conditions. Results confirmed the localization of nanoparticles in the TNTs. Finally, the blood-brain barrier permeability of nanoparticles was measured in vitro in a transwell system and the results showed that discoidal nanoparticles displayed the highest endothelial permeability (~ 1.4x10-5 cm/min) with respect to the other nanoparticles tested. These results make TNTs promising tools for the delivery of drug-loaded discoidal nanoparticles between close and distant cells. This potential is relevant because communication modalities play key roles in driving GBM therapy resistance. Since the formation of TNTs occur also in other type of tumours, these findings can be also exploited in other context.
References
[1] Pinto G, “Patient-derived glioblastoma stem cells transfer mitochondria through tunneling nanotubes in tumor organoids”, Biochem J., vol. 478, no. 1, pp. 21-39, 2021.
[2] Vollmann-Zwerenz A, “Tumor Cell Invasion in Glioblastoma”, Int J Mol Sci, vol. 21, no. 6, pp. 1932, 2020.
[3] Taiarol L, “Givinostat-Liposomes: Anti-Tumor Effect on 2D and 3D Glioblastoma Models and Pharmacokinetics”, Cancers, vol. 14, no. 12, pp. 2978, 2022
Defined Transfer of Colloidal Particles by Electrochemical Microcontact Printing
Full text linkSoft lithography, in particular microcontact printing (µCP), represents a well-established and widespread class of lithographic patterning techniques. It is based on a directed deposition of molecules or colloidal particles by a transfer process with a micro-structured stamp. A critical aspect of µCP is the adhesion cascade that enables the directed transfer of the objects. Here, the interfacial properties of a µCP-stamp are tuned electrochemically to modify the adhesion cascade. During the printing process, the µCP-stamp is submerged in an electrolyte solution and acted as a working electrode whose surface properties depended on the externally applied potential, thus enabling in situ rapid switching of its adhesion properties. As a proof of principle, defined particle patterns are selectively removed from a monolayer of colloidal particles. The adhesion at the particle/solid interface and the transfer mechanisms are determined by using the colloidal probe technique based on atomic force microscopy (AFM). In this case, a single particle is brought into contact with an electrode with the same surface chemistry as the µCP-stamp. Hence, it became possible to determine the electrochemical potential ranges suitable to establish an adhesion cascade