Amorphous Ca²âº polyphosphate nanoparticles regulate the ATP level in bone-like SaOS-2 cells.

Polyphosphate (polyP) is a physiologically occurring polyanion that is synthesized especially in bone-forming osteoblast cells and blood platelets. We used amorphous polyP nanoparticles, complexed with Ca(2+), that have a globular size of ∼100 nm. Because polyP comprises inorganic orthophosphate units that are linked together through high-energy phosphoanhydride bonds, we questioned whether the observed morphogenetic effect, elicited by polyP, is correlated with the energy-generating machinery within the cells. We show that exposure of SaOS-2 osteoblast-like cells to polyP results in a strong accumulation of mitochondria and a parallel translocation of the polyP-degrading enzyme alkaline phosphatase to the cell surface. If SaOS-2 cells are activated by the mineralization activation cocktail (comprising ß-glycerophosphate, ascorbic acid and dexamethasone) and additionally incubated with polyP, a tenfold intracellular increase of the ATP level occurs. Even more, in those cells, an intensified release of ATP into the extracellular space is also seen. We propose and conclude that polyP acts as metabolic fuel after the hydrolytic cleavage of the phosphoanhydride linkages, which contributes to hydroxyapatite formation on the plasma membranes of osteoblasts.

Protein machineries defining pathways of nanocarrier exocytosis and transcytosis.

The transport of nanocarriers through barriers like the gut in a living organism involves the transcytosis of these nanocarriers through the cell layer dividing two compartments. Understanding how this process works is not only essential to further developing strategies for a more effective nanocarrier transport system but also for providing fundamental insights into the barrier function as a means of protection against micro- and nanoplastics in the food chain. We therefore set out to investigate the different uptake mechanisms, intracellular trafficking and the routes for exocytosis for small polystyrene nanoparticles (PS-NPs ca. 100 nm) as mimicking nanocarriers in a Caco-2 cell model for gut-blood transition. We used label-free, quantitative mass spectrometry (MS) for determining the proteome that adhered to transversed nanoparticles. From this rich proteomics dataset, as well as previous studies, we generated stable-transfected Caco-2 cell lines carrying the green fluorescent protein (GFP) coupled to proteins of interest for uptake, early, late and exocytotic endosomes. We detected the spatial and temporal overlap of such marked endosomes with the nanocarrier signal in confocal laser scanning and super-resolution microscopy. There was a clear distinction in the time course of nanoparticle trafficking between groups of proteins for endocytosis, intracellular storage and putatively transcytosis and we identified several key transcytotic markers like Rab3 and Copine1. Moreover, we postulate the necessity of a certain protein composition on endosomes for successful transcytosis of nanocarriers. Finally, we define the two-sided impasse of the lysosome as a dead end for nano-plastic and the limit of nanocarriers in the 100 nm range. STATEMENT OF SIGNIFICANCE: Here we focus on mechanisms of transcytosis and how we can follow these with methods not used before. First, we use mass spectrometry of transcytosed nanoparticles to pick proteins of the transcytosis machinery describing key proteins involved. We can detect the complex mixtures of proteins. As this is a dynamic process involving whole families of proteins interacting with each other and as this is an orchestrated process we coined the term protein machineries for this active interplay. By genetically modifying the proteins attaching GFP we are able to follow the transcytosis pathway. We evaluate the process in a quantitative manner over time. This reveals that the most obvious obstacle to transcytosis is a routing of the nanocarriers to the lysosomes.

Pre-adsorption of antibodies enables targeting of nanocarriers despite a biomolecular corona.

To promote drug delivery to exact sites and cell types, the surface of nanocarriers is functionalized with targeting antibodies or ligands, typically coupled by covalent chemistry. Once the nanocarrier is exposed to biological fluid such as plasma, however, its surface is inevitably covered with various biomolecules forming the protein corona, which masks the targeting ability of the nanoparticle. Here, we show that we can use a pre-adsorption process to attach targeting antibodies to the surface of the nanocarrier. Pre-adsorbed antibodies remain functional and are not completely exchanged or covered by the biomolecular corona, whereas coupled antibodies are more affected by this shielding. We conclude that pre-adsorption is potentially a versatile, efficient and rapid method of attaching targeting moieties to the surface of nanocarriers.

Visualization of the protein corona: towards a biomolecular understanding of nanoparticle-cell-interactions.

The use of nanocarriers in biology and medicine is complicated by the current need to understand how nanoparticles interact in complex biological surroundings. When nanocarriers come into contact with serum, proteins immediately adsorb onto their surface, forming a protein corona which defines their biological identity. Although the composition of the protein corona has been widely determined by proteomics, its morphology still remains unclear. In this study we show for the first time the morphology of the protein corona using transmission electron microscopy. We are able to demonstrate that the protein corona is not, as commonly supposed, a dense, layered shell coating the nanoparticle, but an undefined, loose network of proteins. Additionally, we are now able to visualize and discriminate between the soft and hard corona using centrifugation-based separation techniques together with proteomic characterization. The protein composition of the ∼15 nm hard corona strongly depends on the surface chemistry of the respective nanomaterial, thus further affecting cellular uptake and intracellular trafficking. Large diameter protein corona resulting from pre-incubation with soft corona or Apo-A1 inhibits cellular uptake, confirming the stealth-effect mechanism. In summary, the knowledge on protein corona formation, composition and morphology is essential to design therapeutic effective nanoparticle systems.

Core/shell protein-reactive nanogels via a combination of RAFT polymerization and vinyl sulfone postmodification.

AIM: A promising nanogel vaccine platform was expanded toward antigen conjugation. MATERIALS & METHODS: Block copolymers containing a reactive ester solvophobic block and a PEG-like solvophilic block were synthesized via reversible addition-fragmentation chain-transfer polymerization. Following self-assembly in DMSO, the esters allow for core-crosslinking and hydrophilization by amide bond formation with primary amines. Free thiols were accessed at the polymer chain ends through aminolysis of the reversible addition-fragmentation chain-transfer groups, and into the nanogel core by reactive ester conversion with cysteamine. Subsequently, free thiols were converted into vinyl sulfone moieties. RESULTS: Despite sterical constraints, nanogel-associated vinyl sulfone moieties remained well accessible for cysteins to enforce protein conjugation successfully. CONCLUSION: Our present findings provide a next step toward well-defined vaccine nanoparticles that can co-deliver antigen and a molecular adjuvant.