Markus B. Bannwarth

Mass Spectrometry and Imaging Analysis of Nanoparticle-Containing Vesicles Provide a Mechanistic Insight into Cellular Trafficking

Rational design of nanocarriers for drug delivery approaches requires an unbiased knowledge of uptake mechanisms and intracellular trafficking pathways. Here we dissected these processes using a quantitative proteomics approach. We isolated intracellular vesicles containing superparamagnetic iron oxide polystyrene nanoparticles and analyzed their protein composition by label-free quantitative mass spectrometry. The proteomic snapshot of organelle marker proteins revealed that an atypical macropinocytic-like mechanism mediated the entry of nanoparticles. We show that the entry mechanism is controlled by actin reorganization, atypical macropinocytic signaling, and ADP-ribosylation factor 1. Additionally, our proteomics data demonstrated a central role for multivesicular bodies and multilamellar lysosomes in trafficking and final nanoparticle storage. This was confirmed by confocal microscopy and cryo-TEM measurements. By quantitatively analyzing the protein composition of nanoparticle-containing vesicles, our study clearly defines the routes of nanoparticle entry, intracellular trafficking, and the proteomic milieu of a nanoparticle-containing vesicle.

Well‐Defined Nanofibers with Tunable Morphology from Spherical Colloidal Building Blocks

From particles to fibers: Nanofibers with different morphologies and periodicities can be fabricated by supraparticular assembly of magnetic spherical nanoparticles. A linear sintering process is used to merge the assembled colloids together. The structure of the obtained fibers is controlled by the process parameters and the morphology of the spherical colloidal building blocks.

Colloidal Polymers with Controlled Sequence and Branching Constructed from Magnetic Field Assembled Nanoparticles

The assembly of nanoparticles into polymer-like architectures is challenging and usually requires highly defined colloidal building blocks. Here, we show that the broad size-distribution of a simple dispersion of magnetic nanocolloids can be exploited to obtain various polymer-like architectures. The particles are assembled under an external magnetic field and permanently linked by thermal sintering. The remarkable variety of polymer-analogue architectures that arises from this simple process ranges from statistical and block copolymer-like sequencing to branched chains and networks. This library of architectures can be realized by controlling the sequencing of the particles and the junction points via a size-dependent self-assembly of the single building blocks.

Drug delivery without nanoparticle uptake: delivery by a kiss-and-run mechanism on the cell membrane

Nearly all concepts of nanocarriers as drug delivery devices rely on intracellular uptake. Instead, we demonstrate an alternative concept for rapid and specific delivery of cargo by nanoparticles to TIP47+/ADRP+ lipid droplets. The model can serve as a novel strategy for the non-invasive delivery of drugs by releasing hydrophobic cargo, in our case a model dye, through a kiss-and-run mechanism between nanoparticles and the cell membrane.

Combining the best of two worlds: nanoparticles and nanofibers.

The preparation and applications of nanoparticles and nanofibers are widely described in the literature. Both types of materials have specific advantages but also drawbacks. We discuss here the methods to fabricate nanofibers from nanoparticles and vice versa by template-free methods and colloid-electrospinning. Nanoparticles and nanofibers can be also synergistically combined to yield nanostructured constructs that display highly advantageous properties such as good mechanical integrity, double protection of encapsulated substances, or the possibility to co-encapsulate payloads with different polarities.

Tailor‐Made Nanocontainers for Combined Magnetic‐Field‐Induced Release and MRI

The synthesis of a novel nanocapsule-based carrier system is described, possessing a triggered release in remote-controlled fashion upon application of an external magnetic field in combination with the possibility to use the capsules as contrast agents for magnetic resonance imaging (MRI). Therefore, polymeric nanocontainers containing a high amount of superparamagnetic MnFe2 O4 nanoparticles and a thermo-degradable shell are fabricated via a miniemulsion route. The process allows the facile encapsulation of hydrophilic compounds, as demonstrated for a model dye. Release of the encapsulated dye is achieved upon application of an external alternating magnetic field. While the magnetic nanoparticles here act as heat generators to stimulate the decomposition of the shell and subsequently a release of the payload, they additionally enable the use of the nanocapsules as imaging agents for MRI. Due to the encapsulated magnetic nanoparticles, the nanocapsules possess high r2 relaxivity values of 96-120 Hz mmol(-1) , which makes them suitable for MRI. In toxicity experiments, the nanocapsules show no cell toxicity up to fairly high concentrations (600 µg mL(-1) ). Due to their dual-functionality, the nanocapsules possess high potential as nanocarriers with combined magnetic-field-induced release capability and as contrast agents for MRI.

Iron-loaded PLLA nanoparticles as highly efficient intracellular markers for visualization of mesenchymal stromal cells by MRI

Monitoring of the fate of cells after injection appears paramount for the further development of cell therapies. In this context magnetic resonance imaging (MRI) is increasing in relevance owing to its unique tissue visualization properties. For assessment of cell trafficking and homing, the cells have to be labeled to become MR visible. The rather low sensitivity of MRI demands dedicated intracellular markers with high payloads of MR contrast agents for ensuring sensitive detection of local cell aggregations. In the presented work the application of custom-designed nanometer-sized iron oxide loaded poly-(l-lactide) (iPLLA) nanoparticles was investigated. The particles were synthesized by the mini-emulsion process and evaluated for labeling of mesenchymal stromal cells (MSCs). The efficient cellular uptake and long intracellular retention times of the particles as well as their nontoxicity are demonstrated. The average cellular iron content was 55 pg iron per cell. Further incorporation of, for example, fluorescent dye enables the generation of multireporter particles, providing the great potential for multimodal imaging. The efficiency of these nanoparticles as MRI contrast agent was evaluated in vitro using relaxation rate mapping, yielding relaxivities r2 = 273.3, r2 (*) = 545.1 mm(-1) s(-1) at 3 T and r2 = 415.7, r2 (*) = 872.3 mm(-1) s(-1) at 11.7 T. The high r2 (*) relaxivity of the iPLLA nanoparticles enabled visualization of a single labeled cell in vitro at 50-µm spatial resolution. In vivo evaluation in a rat injury model revealed the potential of the iPLLA particles to efficiently label MSCs for MRI monitoring of ~20 000-40 000 injected cells at 11.7 T. In conclusion the presented work demonstrates the applicability of iPLLA particles as efficient intracellular marker for MSC labeling for monitoring the fate of the cells by MRI.

Preparation of ellipsoid-shaped supraparticles with modular compositions and investigation of shape-dependent cell-uptake

Hybrid colloidal supraparticles often show a superior performance in catalysis, optics and biomedicine thanks to the synergistic effect of the ensemble of their single nanoparticle building blocks. Despite the emerging importance of shape-dependent properties of nanostructures, the synthesis of supraparticles is generally limited to a spherical shape. Here, a broadly applicable method is presented for the fabrication of ellipsoid supraparticles from one or several types of inorganic nanoparticles in various compositions. The method is highly versatile and modular, allowing free choice of hydrophobic nanoparticles to combine desired properties in the resulting supraparticles. A representative series of ellipsoid-shaped supraparticles is fabricated and their morphology, hybrid structure and composition as well as their functional properties are investigated. All employed nanoparticle types are successfully incorporated resulting in ellipsoid-shaped supraparticles with largely homogeneous intra- and interparticular distribution of the different nanoparticle building blocks. A biological assessment of iron oxide ellipsoid supraparticles reveals no safety issues but a pronounced lower cellular uptake compared to spherical ones. This distinct shape–property relationship illustrates the importance of the supraparticle shape as a parameter for the rational design of nanosystems for biomedical applications.

Monitoring crack appearance and healing in coatings with damage self-reporting nanocapsules

Autonomous highlighting of damage in protective polymer coatings allows on-demand maintenance and enables prolongation of the lifetimes of the coated materials. To monitor the entire cycle of damage occurrence and successful healing, one must be able to visualize both processes and display the current health-state of the coating. Herein, we equipped coatings with nanocapsules that can self-indicate their mechanical micro-damage via color development. Hence, whenever the coating was damaged, the capsules broke and highlighted the damaged spot. As a second feature, the color development was reversed and discoloration occurred in the presence of (self-)healing compounds, allowing the user to monitor the healing process. Thus, in the first step, damages were highlighted via color “turn-on” and in the subsequent step a propagating healing reaction “turns-off” the damage indication system to trace the healing reaction and allow monitoring of the entire health cycle.

Multifunctional clickable and protein-repellent magnetic silica nanoparticles

Silica nanoparticles are versatile materials whose physicochemical surface properties can be precisely adjusted. Because it is possible to combine several functionalities in a single carrier, silica-based materials are excellent candidates for biomedical applications. However, the functionality of the nanoparticles can get lost upon exposure to biological media due to uncontrolled biomolecule adsorption. Therefore, it is important to develop strategies that reduce non-specific protein-particle interactions without losing the introduced surface functionality. Herein, organosilane chemistry is employed to produce magnetic silica nanoparticles bearing differing amounts of amino and alkene functional groups on their surface as orthogonally addressable chemical functionalities. Simultaneously, a short-chain zwitterion is added to decrease the non-specific adsorption of biomolecules on the nanoparticles surface. The multifunctional particles display reduced protein adsorption after incubation in undiluted fetal bovine serum as well as in single protein solutions (serum albumin and lysozyme). Besides, the particles retain their capacity to selectively react with biomolecules. Thus, they can be covalently bio-functionalized with an antibody by means of orthogonal click reactions. These features make the described multifunctional silica nanoparticles a promising system for the study of surface interactions with biomolecules, targeting, and bio-sensing.