Extracellular vesicles from the microalga Tetraselmis chuii are biocompatible and exhibit unique bone tropism along with antioxidant and anti-inflammatory properties.

Extracellular vesicles (EVs) are membrane-enclosed bio-nanoparticles secreted by cells and naturally evolved to transport various bioactive molecules between cells and even organisms. These cellular objects are considered one of the most promising bio-nanovehicles for the delivery of native and exogenous molecular cargo. However, many challenges with state-of-the-art EV-based candidates as drug carriers still exist, including issues with scalability, batch-to-batch reproducibility, and cost-sustainability of the final therapeutic formulation. Microalgal extracellular vesicles, which we named nanoalgosomes, are naturally released by various microalgal species. Here, we evaluate the innate biological properties of nanoalgosomes derived from cultures of the marine microalgae Tetraselmis chuii, using an optimized manufacturing protocol. Our investigation of nanoalgosome biocompatibility in preclinical models includes toxicological analyses, using the invertebrate model organism Caenorhabditis elegans, hematological and immunological evaluations ex vivo and in mice. We evaluate nanoalgosome cellular uptake mechanisms in C. elegans at cellular and subcellular levels, and study their biodistribution in mice with accurate space-time resolution. Further examination highlights the antioxidant and anti-inflammatory bioactivities of nanoalgosomes. This holistic approach to nanoalgosome functional characterization demonstrates that they are biocompatible and innate bioactive effectors with unique bone tropism. These findings suggest that nanoalgosomes have significant potential for future therapeutic applications.

Interfacial Denaturation at the Droplet Simplifies the Formation of Drug-Loaded Protein Nanocapsules to Enhance Immune Response of Cells.

Nanocapsules enable multicomponent encapsulation of therapeutic cargoes with high encapsulation content and efficiency, which is vital for cancer immunotherapy. In the past, chemical crosslinking is used to synthesize nanocapsules, which can impede the regulatory approval process. Therefore, a new class of protein nanocapsules is developed by eliminating the need for chemical crosslinking by utilizing protein denaturation through a process that is referred to as "baking at the droplet interface". Such protein nanocapsules with antigens incorporated in the shell and a combination of encapsulated drugs showed an enhancement in the immune response of cells.

Single-Atom Catalysts through Pressure-Controlled Metal Diffusion.

Single-atom catalysts (SACs) open up new possibilities for advanced technologies. However, a major complication in preparing high-density single-atom sites is the aggregation of single atoms into clusters. This complication stems from the delicate balance between the diffusion and stabilization of metal atoms during pyrolysis. Here, we present pressure-controlled metal diffusion as a new concept for fabricating ultra-high-density SACs. Reducing the pressure inhibits aggregation substantially, resulting in almost three times higher single-atom loadings than those obtained at ambient pressure. Molecular dynamics and computational fluid dynamics simulations reveal the role of a metal hopping mechanism, maximizing the metal atom distribution through an increased probability of metal-ligand binding. The investigation of the active site density by electrocatalytic oxygen reduction validates the robustness of our approach. The first realization of Ullmann-type carbon-oxygen couplings catalyzed on single Cu sites demonstrates further options for efficient heterogeneous catalysis.

In Situ Measurement of Nanoparticle-Blood Protein Adsorption and Its Heterogeneity with Single-Nanoparticle Resolution via Dual Fluorescence Quantification.

The formation of a protein corona gives nanomedicines a distinct biological identity, profoundly influencing their fate in the body. Nonspecific nanoparticle-protein interactions are typically highly heterogeneous, which can lead to unique biological behaviors and in vivo fates for individual nanoparticles that remain underexplored. To address this, we have established an in situ approach that allows quantitative examination of nanoparticle-protein adsorption at the individual nanoparticle level. This method integrates dual fluorescence quantification techniques, wherein the nanoparticles are first individually analyzed via nanoflow cytometry to detect fluorescent signals from adsorbed proteins. The obtained fluorescence intensity is then translated into protein quantities through calibration with microplate reader quantification. Consequently, this approach enables analysis of interparticle heterogeneity of nano-protein interactions, as well as in situ monitoring of protein adsorption kinetics and nanoparticle aggregation status in blood serum, preconditioning for a comprehensive understanding of nano-bio interactions, and predicting in vivo fate of nanomedicines.

Size-Dependent Photocatalytic Reactivity of Conjugated Microporous Polymer Nanoparticles.

Particle size is a critical factor for improving photocatalytic reactivity of conjugated microporous polymers (CMPs) as mass transfer in the porous materials is often the rate-limiting step. However, due to the synthetic challenge of controlling the size of CMPs, the impact of particle size is yet to be investigated. To address this problem, a simple and versatile dispersion polymerization route that can synthesize dispersible CMP nanoparticles with controlled size from 15 to 180 nm is proposed. Leveraging the precise control of the size, it is demonstrated that smaller CMP nanoparticles have dramatically higher photocatalytic reactivity in various organic transformations, achieving more than 1000% enhancement in the reaction rates by decreasing the size from 180 to 15 nm. The size-dependent photocatalytic reactivity is further scrutinized using a kinetic model and transient absorption spectroscopy, revealing that only the initial 5 nm-thick surface layer of CMP nanoparticles is involved in the photocatalytic reactions because of internal mass transfer limitations. This finding substantiates the potential of small CMP nanoparticles to efficiently use photo-generated excitons and improve energy-efficiency of numerous photocatalytic reactions.

Ion and Molecular Sieving With Ultrathin Polydopamine Nanomembranes.

In contrast to biological cell membranes, it is still a major challenge for synthetic membranes to efficiently separate ions and small molecules due to their similar sizes in the sub-nanometer range. Inspired by biological ion channels with their unique channel wall chemistry that facilitates ion sieving by ion-channel interactions, the first free-standing, ultrathin (10-17 nm) nanomembranes composed entirely of polydopamine (PDA) are reported here as ion and molecular sieves. These nanomembranes are obtained via an easily scalable electropolymerization strategy and provide nanochannels with various amine and phenolic hydroxyl groups that offer a favorable chemical environment for ion-channel electrostatic and hydrogen bond interactions. They exhibit remarkable selectivity for monovalent ions over multivalent ions and larger species with K+/Mg2+ of ≈4.2, K+/[Fe(CN)6]3- of ≈10.3, and K+/Rhodamine B of ≈273.0 in a pressure-driven process, as well as cyclic reversible pH-responsive gating properties. Infrared spectra reveal hydrogen bond formation between hydrated multivalent ions and PDA, which prevents the transport of multivalent ions and facilitates high selectivity. Chemically rich, free-standing, and pH-responsive PDA nanomembranes with specific interaction sites are proposed as customizable high-performance sieves for a wide range of challenging separation requirements.

Supramolecular Assembly in Live Cells Mapped by Real-Time Phasor-Fluorescence Lifetime Imaging.

The complex dynamics and transience of assembly pathways in living systems complicate the understanding of these molecular to nanoscale processes. Current technologies are unable to track the molecular events leading to the onset of assembly, where real-time information is imperative to correlate their rich biology. Using a chemically designed pro-assembling molecule, we map its transformation into nanofibers and their fusion with endosomes to form hollow fiber clusters. Tracked by phasor-fluorescence lifetime imaging (phasor-FLIM) in epithelial cells (L929, A549, MDA-MB 231) and correlative light-electron microscopy and tomography (CLEM), spatiotemporal splicing of the assembly events shows time-correlated metabolic dysfunction. The biological impact begins with assembly-induced endosomal disruption that reduces glucose transport into the cells, which, in turn, stymies mitochondrial respiration.

Triazine Frameworks for the Photocatalytic Selective Oxidation of Toluene.

Investigations into the selective oxidation of inert sp3 C-H bonds using polymer photocatalysts under mild conditions have been limited. Additionally, the structure-activity relationship of photocatalysts often remains insufficiently explored. Here, a series of thiophene-based covalent triazine frameworks (CTFs) are used for the efficient and selective oxidation of hydrocarbons to aldehydes or ketones under ambient aerobic conditions. Spectroscopic methods conducted in situ and density functional theory (DFT) calculations revealed that the sulfur atoms within the thiophene units play a pivotal role as oxidation sites due to the generation of photogenerated holes. The effect of photogenerated holes on photocatalytic toluene oxidation was investigated by varying the length of the spacer in a CTF donor-acceptor based photocatalyst. Furthermore, the manipulation of reactive oxygen species was employed to enhance selectivity by weakening the peroxidative capacity. As an illustrative example, this study successfully demonstrated the synthesis of a precursor of the neurological drug AMG-579 using a photocatalytic protocol.

Dipeptide coacervates as artificial membraneless organelles for bioorthogonal catalysis.

Artificial organelles can manipulate cellular functions and introduce non-biological processes into cells. Coacervate droplets have emerged as a close analog of membraneless cellular organelles. Their biomimetic properties, such as molecular crowding and selective partitioning, make them promising components for designing cell-like materials. However, their use as artificial organelles has been limited by their complex molecular structure, limited control over internal microenvironment properties, and inherent colloidal instability. Here we report the design of dipeptide coacervates that exhibit enhanced stability, biocompatibility, and a hydrophobic microenvironment. The hydrophobic character facilitates the encapsulation of hydrophobic species, including transition metal-based catalysts, enhancing their efficiency in aqueous environments. Dipeptide coacervates carrying a metal-based catalyst are incorporated as active artificial organelles in cells and trigger an internal non-biological chemical reaction. The development of coacervates with a hydrophobic microenvironment opens an alternative avenue in the field of biomimetic materials with applications in catalysis and synthetic biology.

Intrinsic Burst-Blinking Nanographenes for Super-Resolution Bioimaging.

Single-molecule localization microscopy (SMLM) is a powerful technique to achieve super-resolution imaging beyond the diffraction limit. Although various types of blinking fluorophores are currently considered for SMLM, intrinsic blinking fluorophores remain rare at the single-molecule level. Here, we report the synthesis of nanographene-based intrinsic burst-blinking fluorophores for highly versatile SMLM. We image amyloid fibrils in air and in various pH solutions without any additive and lysosome dynamics in live mammalian cells under physiological conditions. In addition, the single-molecule labeling of nascent proteins in primary sensory neurons was achieved with azide-functionalized nanographenes via click chemistry. SMLM imaging reveals higher local translation at axonal branching with unprecedented detail, while the size of translation foci remained similar throughout the entire network. These various results demonstrate the potential of nanographene-based fluorophores to drastically expand the applicability of super-resolution imaging.

Effect of Protein Corona on the Specificity and Efficacy of Nanobioconjugates to Treat Intracellular Infections.

Encapsulating drugs into functionalized nanoparticles (NPs) is an alternative to reach the specific therapeutic target with lower doses. However, when the NPs are in contact with physiological media, proteins adsorb on their surfaces, forming a protein corona (PC) biomolecular layer, acquiring a distinct biological identity that alters their interactions with cells. Itraconazole (ITZ), an antifungal agent, is encapsulated into PEGylated and/or functionalized NPs with high specificity for macrophages. It is evaluated how the PC impacts their cell uptake and antifungal effect. The minimum inhibitory concentration and colony-forming unit assays demonstrate that encapsulated ITZ into poly(ethylene glycol) (PEG) NPs improves the antifungal effect compared with NPs lacking PEGylation. The improvement can be related to the synergistic effect of the encapsulated ITZ and NPs composition and the reduction of PC formation in PEG NPs. Functionalized NPs with anti-F4/80 and anti-MARCO antibodies, or mannose without PEG and treated with PC, show an improved uptake but, in the presence of PEG, significantly reduce the endocytosis, dominating the stealth effect from PEG. Therefore, the PC plays a crucial role in the nanosystem uptake and antifungal effects, which suggests the need for in vivo model studies to evaluate the effect of PC in the specificity and biodistribution.

Uptake of extracellular vesicles into immune cells is enhanced by the protein corona.

The influence of a protein corona on the uptake of nanoparticles in cells has been demonstrated in various publications over the last years. Extracellular vesicles (EVs), can be seen as natural nanoparticles. However, EVs are produced under different cell culture conditions and little is known about the protein corona forming on EVs and its influence on their uptake by target cells. Here, we use a proteomic approach in order to analyze the protein composition of the EVs themselves and the protein composition of a human blood plasma protein corona around EVs. Moreover, we analyze the influence of the protein corona on EV uptake into human monocytes and compare it with the influence on the uptake of engineered liposomes. We show that the presence of a protein corona increases the uptake of EVs in human monocytes. While for liposomes this seems to be triggered by the presence of immunoglobulins in the protein corona, for EVs blocking the Fc receptors on monocytes did not show an influence of uptake. Therefore, other mechanisms of docking to the cell membrane and uptake are most like involved, demonstrating a clear difference between EVs and liposomes as technically produced nanocarriers.

Polymeric Microreactors with pH-Controlled Spatial Localization of Cascade Reactions.

Lipid and polymer vesicles provide versatile means of creating systems that mimic the architecture of cells. However, these constructs cannot mimic the adaptive compartmentalization observed in cells, where the assembly and disassembly of subcompartments are dynamically modulated by environmental cues. Here, we describe a fully polymeric microreactor with a coacervate-in-vesicle architecture that exhibits an adaptive response to pH. The system was fabricated by microfluidic generation of semipermeable biomimetic polymer vesicles within 1 min using oleyl alcohol as the oil phase. The polymersomes allowed for the diffusion of protons and substrates acting as external signals. Using this method, we were able to construct adaptive microreactors containing internal polyelectrolyte-based catalytic organelles capable of sequestering and localizing enzymes and reaction products in a dynamic process driven by an external stimulus. This approach provides a platform for the rapid and efficient construction of robust adaptive microreactors that can be used in catalysis, biosensing, and cell mimicry.

Proteomics reveals time-dependent protein corona changes in the intracellular pathway.

The intracellular protein corona has not been fully investigated in the field of nanotechnology-biology (nano-bio) interactions. To effectively understand intracellular protein corona formation and dynamics, we established a workflow to isolate the intracellular protein corona at different uptake times of two nanoparticles - magnetic hydroxyethyl starch nanoparticles (HES-NPs) and magnetic human serum albumin nanocapsules (HSA-NCs). We performed label-free quantitative LC-MS proteomics to analyze the composition of the intracellular protein corona and correlated our findings with results from conventional methods for intracellular trafficking of nanocarriers, such as flow cytometry, transmission electron microscopy (TEM), and confocal microscopy (cLSM). We determined the evolution of the intracellular protein corona. At different time stages the protein corona of the HES-NPs with a slower uptake changed, but there were fewer changes in that of the HSA-NCs with a more rapid uptake. We identified proteins that are involved in macropinocytosis (RAC1, ASAP2) as well as caveolin. This was confirmed by blocking experiments and by TEM studies. The investigated nanocarrier predominantly trafficked from early endosomes as determined by RAB5 identification in proteomics and in cLSM to late endosomes/lysosomes (RAB7, LAMP1, cathepsin K and HSP 90-beta) We further demonstrated differences between nanoparticles with slower and faster uptake kinetics and determined the associated proteome at different time points. Analysis of the intracellular protein corona provides us with effective data to examine the intracellular trafficking of nanocarriers used in efficient drug delivery and intracellular applications. STATEMENT OF SIGNIFICANCE: Many research papers focus on the protein corona on nanoparticles formed in biological fluids, but there are hardly any articles dealing with proteins that come in contact with nanoparticles inside cells. The "intracellular protein corona" studied here is a far more complex and highly demanding field. Most nanocarriers are designed to be taken up into cells. Given this, we chose two different nanocarriers to reveal changes in the proteins in dendritic cells during contact at specific times. Further studies will allow us to examine molecular target proteins using these methods. Our research is a significant addition towards the goal of understanding and thus improving the efficacy of drug nanocarriers.

Anti-PEG antibodies enriched in the protein corona of PEGylated nanocarriers impact the cell uptake.

Poly(ethylene glycol) (PEG) is the gold standard used to reduce unspecific protein adsorption and prolong nanocarrier circulation time. However, this stealth effect could be counteracted by the increasing prevalence of anti-PEG antibodies in the bloodstream. Up to now, the presence of anti-PEG antibodies in the protein corona and their effect on cell uptake has not been investigated yet. Our results showed a high concentration and prevalence of anti-PEG antibodies in the German population. PEGylated nanocarriers exhibited a higher level of anti-PEG antibodies in the protein corona compared to non-PEGylated, which lead to higher uptake in macrophages. Consequently, the anti-PEG antibodies in the protein corona could mitigate the stealth effect of PEG, leading to accelerated blood clearance and unwanted side effects.

Delivery of Immunostimulatory Cargos in Nanocarriers Enhances Anti-Tumoral Nanovaccine Efficacy.

Finding a long-term cure for tumor patients still represents a major challenge. Immunotherapies offer promising therapy options, since they are designed to specifically prime the immune system against the tumor and modulate the immunosuppressive tumor microenvironment. Using nucleic-acid-based vaccines or cellular vaccines often does not achieve sufficient activation of the immune system in clinical trials. Additionally, the rapid degradation of drugs and their non-specific uptake into tissues and cells as well as their severe side effects pose a challenge. The encapsulation of immunomodulatory molecules into nanocarriers provides the opportunity of protected cargo transport and targeted uptake by antigen-presenting cells. In addition, different immunomodulatory cargos can be co-delivered, which enables versatile stimulation of the immune system, enhances anti-tumor immune responses and improves the toxicity profile of conventional chemotherapeutic agents.

Aggregation induced nucleic acids recognition by homodimeric asymmetric monomethyne cyanine fluorochromes in mesenchymal stem cells.

In the light of recent retrovirus pandemics, the issue of discovering new and diverse RNA-specific fluorochromes for research and diagnostics became of acute importance. The great majority of nucleic acid-specific probes either do not stain RNA or cannot distinguish between DNA and RNA. The versatility of polymethine dyes makes them suitable as stains for visualization, analysis, and detection of nucleic acids, proteins, and other biomolecules. We synthesized the asymmetric dicationic homodimeric monomethine cyanine dyes 1,1'-(1,3-phenylenebis(methylene))bis(4-((3-methylbenzo[d]thiazol-2(3H)-ylidene)methyl)pyridin-1-ium) bromide (Т1) and 1,1'-(1,3-phenylenebis(methylene))bis(4-((3-methylbenzo[d]thiazol-2(3H)-ylidene)methyl)quinolin-1-ium) bromide (M1) and tested their binding specificity, spectral characteristics, membrane penetration in living and fixed cells, cellular toxicity, and stability of fluorescent emission. Mesenchymal cells have diverse phenotypes and extensive proliferation and differentiation properties. We found dyes T1 and M1 to show high photochemical stability in living mesenchymal stem cells from apical papilla (SCAP) with a strong fluorescent signal when bound to nucleic acids. We found M1 to perform better than control fluorochrome (Hoechst 33342) for in vivo DNA visualization. T1, on the other hand, stains granular cellular structures resembling ribosomes in living cells and after permeabilization of the nuclear membrane stains the nucleoli and not the chromatin in the nucleus. This makes T1 suitable for the visualization of structures rich in RNA in living and fixed cells.

Liposomal Enzyme Nanoreactors Based on Nanoconfinement for Efficient Antitumor Therapy.

Enzymatic reactions can consume endogenous nutrients of tumors and produce cytotoxic species and are therefore promising tools for treating malignant tumors. Inspired by nature where enzymes are compartmentalized in membranes to achieve high reaction efficiency and separate biological processes with the environment, we develop liposomal nanoreactors that can perform enzymatic cascade reactions in the aqueous nanoconfinement of liposomes. The nanoreactors effectively inhibited tumor growth in vivo by consuming tumor nutrients (glucose and oxygen) and producing highly cytotoxic hydroxyl radicals (⋅OH). Co-compartmentalization of glucose oxidase (GOx) and horseradish peroxidase (HRP) in liposomes could increase local concentration of the intermediate product hydrogen peroxide (H2 O2 ) as well as the acidity due to the generation of gluconic acid by GOx. Both H2 O2 and acidity accelerate the second-step reaction by HRP, hence improving the overall efficiency of the cascade reaction. The biomimetic compartmentalization of enzymatic tandem reactions in biocompatible liposomes provides a promising direction for developing catalytic nanomedicines in antitumor therapy.