Systematic screening of different polyglycerin‐based dienophile macromonomers for efficient nanogel formation through IEDDA inverse nanoprecipitation

Alternatives for strain‐promoted azide–alkyne cycloaddition (SPAAC) chemistries are needed because of the employment of expensive and not easily scalable precursors such as bicyclo[6.1.0]non‐4‐yne (BCN). Inverse electron demand Diels Alder (iEDDA)‐based click chemistries, using dienophiles and tetrazines, offer a more bioorthogonal and faster toolbox, especially in the biomedical field. Here, the straightforward synthesis of dendritic polyglycerin dienophiles (dPG‐dienophiles) and dPG‐methyl‐tetrazine (dPG‐metTet) as macromonomers for a fast, stable, and scalable nanogel formation by inverse nanoprecipitation is reported. Nanogel size–influencing parameters are screened such as macromonomer concentration and water‐to‐acetone ratio are screened. dPG‐norbonene and dPG‐cyclopropene show fast and stable nanogel formation in the size range of 40–200 nm and are thus used for the coprecipitation of the model protein myoglobin. High encapsulation efficiencies of more than 70% at a 5 wt% feed ratio are obtained in both cases, showing the suitability of the mild gelation chemistry for the encapsulation of small proteins

Polyglycerin-Based Nanogels for Protein Encapsulation

Smart and sensitive nanocarriers for the delivery of therapeutic proteins are needed as alternatives for covalent modification with the potentially immunogenic PEG. Nanogels as water swollen, highly hydrophilic polymer networks are promising candidates for protein delivery vehicles. However, scalable production, under sensitive and mild conditions, is still an active area of research. Inverse nanoprecipitation, as one of several production methods, offers the potential for the mild and non-destructive encapsulation of sensitive proteins. The gel networks are preferably formed by crosslinking of biocompatible, hydrophilic, and easily obtainable functionalized polymers. A variety of crosslinking chemistries, such as CuAAC, Thiol-Michael addition, and SPAAC have been studied for this purpose. Most of these chemistries, however, suffer from low biorthogonality, toxic catalysts, or the low synthetic accessibility of the precursors. IEDDA has emerged as an alternative for the other click chemistries, with fast reaction kinetics, high biorthogonality and easily accessible precursors. The goal of this study was to design nanogels in a way that most of the mentioned criteria for a successful nanocarrier system are fulfilled. Nanogels, based on the biocompatible, scalable, hydrophilic and easily functionalizable dPG were presented in this work. Inverse nanoprecipitation was used as a mild gelation method, that lacks toxic surfactants or damaging ultrasound. The bioorthogonal and fast iEDDA click chemistry, based on tetrazines and dienophiles, was established for the first time in the use of nanogel production. The first study focused on the search for suitable dienophiles for the iEDDA crosslinking chemistry. Reactivity and scalability were most important. This was achieved by screening of different iEDDA-reactive dienophile macromonomers. For this, the four different dienophile macromonomers dPG-norbonene, dPG-BCN, dPG-cyclopropene, and dPG-DHP were synthesized. As the tetrazine counterpart, the stable but still reactive dPG-metTet was obtained. The macromonomers were compared regarding their ability to form macro-and nanogels. Gelation times were determined and revealed that only dPG-norbonene and dPG-cyclopropene were able to form macrogels, while dPG-BCN showed incomplete, and dPG-DHP no gel formation at all. For nanogel formation, reaction parameters, such as rotation speed, macromonomer concentration, quenching times, and solvent to non-solvent ratios were screened. Solvent to non-solvent ratio and quenching time were the most influential parameters on nanogel size and polydispersity. The nanogels were obtained in the relevant size range of 40 to 200 nm and were stable for at least several months in aqueous solution. Co-precipitation of the small model protein myoglobin was performed with the most promising macromonomer candidates dPG-norbonene and -cyclopropene. Encapsulation efficiencies of above 70% were achieved. Thus, it could be shown that a combination of dPG as the polymer scaffold, together with easily obtainable iEDDA reactive groups, such as norbonene and methyl tetrazine provide the toolbox for the design of a scalable and functional nanocarrier for proteins. The second study aimed at transferring the gained knowledge on nanogel formation parameters, such as quenching time and solvent to non-solvent ratio on a smart, environmentally responsive version of the nanogel system. Environmentally responsiveness was achieved by the introduction of pH-cleavable acetal groups. One which is cleavable at pH values below 5 (benzacetal) and one which cleaves at values below 3 (THP). For this dPG was functionalized with the respective acetal linkers and then further functionalized with the dienophiles norbonene and BCN from the first study. Norbonene was the most promising candidate and BCN was used as a well-established comparison. The macromonomers showed no toxicity up to concentrations of 2.5 mg/mL in three different cell lines. Nanogels in the size range of 47-200 nm were obtained, which were stable in aqueous solution at pH 7.4 for several months, without decomposition or an increase of polydispersity. Upon exposure to acidic conditions, the benzacetal-based nanogels cleaved to small particles at pH 4.5 within 48 h, while the THP acetal-based nanogels cleaved only at pH 3 to small particles after 48 h. This proved the applicability of the nanogels for lysosomal cleavage and intracellular delivery for benzacetal gels and a potential delivery to the small intestine by the THP acetal functionalized gels. Co-precipitation of the therapeutic protein asparaginase led to encapsulation efficiencies of up to 93%. The degradability of the gels, the high encapsulation efficiencies, as well as the synthetic accessibility and biocompatibility of the macromonomer precursors, point out the potential of this nanocarrier platform for biomedical applications. Based on the data that was obtained, the potential of the iEDDA based nanogels is evident. However, scalability must be improved at least for the nanogel production itself. Continuous flow methods, such as microfluidic based nanoprecipitation could potentially be used for the upscaling of the nanogels presented in this work. Furthermore, the addition of active targeting ligands to the nanogels or the macromonomers before inverse nanoprecipitation would even further increase the applicability of these nanogels for biomedical applications. One way of an easily obtainable active targeting moiety would be the sulfation of the dPG-macromonomers, which would introduce L-selectin binding affinity into the nanogels, thus targeting inflamed tissues

Synthesis of pH-degradable polyglycerol-based nanogels by iEDDA-mediated crosslinking for encapsulation of asparaginase using inverse nanoprecipitation

Biocompatible, environmentally responsive, and scalable nanocarriers are needed for targeted and triggered delivery of therapeutic proteins. Suitable polymers, preparation methods, and crosslinking chemistries must be considered for nanogel formation. Biocompatible dendritic polyglycerol (dPG) is used in the mild, surfactant-free inverse nanoprecipitation method for nanogel preparation. The biocompatible, fast, and bioorthogonal inverse electron demand Diels-Alder (iEDDA) crosslinking chemistry is used. In this work, the synthesis of pH-degradable nanogels, based on tetrazine, norbonene, and bicyclo[6.1.0]nonyne (BCN) functionalized macromonomers, is reported. The macromonomers are non-toxic up to 2.5 mg mL−1 in three different cell lines. Nanogels are obtained in the size range of 47 to 200 nm and can be degraded within 48 h at pH 4.5 (BA-gels), and pH 3 (THP-gels), respectively. Encapsulation of asparaginase (32 kDa) yield encapsulation efficiencies of up to 93% at 5 wt.% feed. Overall, iEDDA-crosslinked pH-degradable dPG-nanogels from inverse nanoprecipitation are promising candidates for biomedical applications

Topology-Matching Design of an Influenza-Neutralizing Spiky Nanoparticle-Based Inhibitor with a Dual Mode of Action

In this study, we demonstrate the concept of "topology-matching design" for virus inhibitors. With the current knowledge of influenzaA virus (IAV), we designed a nanoparticle-based inhibitor (nano-inhibitor) that has a matched nanotopology to IAV virions and shows heteromultivalent inhibitory effects on hemagglutinin and neuraminidase. The synthesized nano-inhibitor can neutralize the viral particle extracellularly and block its attachment and entry to the host cells. The virus replication was significantly reduced by 6 orders of magnitude in the presence of the reverse designed nano-inhibitors. Even when used 24hours after the infection, more than 99.999% inhibition is still achieved, which indicates such a nano-inhibitor might be a potent antiviral for the treatment of influenza infection

Graphene-Assisted Synthesis of 2D Polyglycerols as Innovative Platforms for Multivalent Virus Interactions

2D nanomaterials have garnered widespread attention in biomedicine and bioengineering due to their unique physicochemical properties. However, poor functionality, low solubility, intrinsic toxicity, and nonspecific interactions at biointerfaces have hampered their application in vivo. Here, biocompatible polyglycerol units are crosslinked in two dimensions using a graphene-assisted strategy leading to highly functional and water-soluble polyglycerols nanosheets with 263 +/- 53 nm and 2.7 +/- 0.2 nm average lateral size and thickness, respectively. A single-layer hyperbranched polyglycerol containing azide functional groups is covalently conjugated to the surface of a functional graphene template through pH-sensitive linkers. Then, lateral crosslinking of polyglycerol units is carried out by loading tripropargylamine on the surface of graphene followed by lifting off this reagent for an on-face click reaction. Subsequently, the polyglycerol nanosheets are detached from the surface of graphene by slight acidification and centrifugation and is sulfated to mimic heparin sulfate proteoglycans. To highlight the impact of the two-dimensionality of the synthesized polyglycerol sulfate nanosheets at nanobiointerfaces, their efficiency with respect to herpes simplex virus type 1 and severe acute respiratory syndrome corona virus 2 inhibition is compared to their 3D nanogel analogs. Four times stronger in virus inhibition suggests that 2D polyglycerols are superior to their current 3D counterparts

Graphene‐Assisted Synthesis of 2D Polyglycerols as Innovative Platforms for Multivalent Virus Interactions

2D nanomaterials have garnered widespread attention in biomedicine and bioengineering due to their unique physicochemical properties. However, poor functionality, low solubility, intrinsic toxicity, and nonspecific interactions at biointerfaces have hampered their application in vivo. Here, biocompatible polyglycerol units are crosslinked in two dimensions using a graphene-assisted strategy leading to highly functional and water-soluble polyglycerols nanosheets with 263 ± 53 nm and 2.7 ± 0.2 nm average lateral size and thickness, respectively. A single-layer hyperbranched polyglycerol containing azide functional groups is covalently conjugated to the surface of a functional graphene template through pH-sensitive linkers. Then, lateral crosslinking of polyglycerol units is carried out by loading tripropargylamine on the surface of graphene followed by lifting off this reagent for an on-face click reaction. Subsequently, the polyglycerol nanosheets are detached from the surface of graphene by slight acidification and centrifugation and is sulfated to mimic heparin sulfate proteoglycans. To highlight the impact of the two-dimensionality of the synthesized polyglycerol sulfate nanosheets at nanobiointerfaces, their efficiency with respect to herpes simplex virus type 1 and severe acute respiratory syndrome corona virus 2 inhibition is compared to their 3D nanogel analogs. Four times stronger in virus inhibition suggests that 2D polyglycerols are superior to their current 3D counterparts.Peer Reviewe

Streaming Live Neuronal Simulation Data into Visualization and Analysis

Neuroscientists want to inspect the data their simulations are producing while these are still running. This will on the one hand save them time waiting for results and therefore insight. On the other, it will allow for more efficient use of CPU time if the simulations are being run on supercomputers. If they had access to the data being generated, neuroscientists could monitor it and take counter-actions, e.g., parameter adjustments, should the simulation deviate too much from in-vivo observations or get stuck.As a first step toward this goal, we devise an in situ pipeline tailored to the neuroscientific use case. It is capable of recording and transferring simulation data to an analysis/visualization process, while the simulation is still running. The developed libraries are made publicly available as open source projects. We provide a proof-of-concept integration, coupling the neuronal simulator NEST to basic 2D and 3D visualization.KeywordsNeuroscientific simulation In situ visualizatio

Streaming Live NEST Simulation Data Into Visualization and Analysis

Neuroscientists want to inspect the data their simulations are producing while these are still running. This will on the one hand save them time waiting for results and therefore insight. On the other, it will allow for more efficient use of CPU time if the simulations are being run on supercomputers. If they had access to the data being generated, neuroscientists could monitor it and take counter-actions, e.g., parameter adjustments, should the simulation deviate too much from in-vivo observations or get stuck.As a first step toward this goal, we devise an in situ pipeline tailored to the neuroscientific use case. It is capable of recording and transferring simulation data to an analysis/visualization process, while the simulation is still running. The developed libraries are made publicly available as open source projects. We provide a proof-of-concept integration, coupling the neuronal simulator NEST to basic 2D and 3D visualization