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Extracellular vesicles (EVs) are highly interesting for the design of next-generation therapeutics. However, their preparation methods face challenges in standardization, yield, and reproducibility. Here, we describe a highly efficient and reproducible EV preparation method for monodisperse nano plasma membrane vesicles (nPMVs), which yields 10 to 100 times more particles per cell and hour than conventional EV preparation methods. nPMVs are produced by homogenizing giant plasma membrane vesicles following cell membrane blebbing and apoptotic body secretion induced by chemical stressors. nPMVs showed no significant differences compared to native EVs from the same cell line in cryo-TEM analysis, in vitro cellular interactions, and in vivo biodistribution studies in zebrafish larvae. Proteomics and lipidomics, on the other hand, suggested substantial differences consistent with the divergent origin of these two EV types and indicated that nPMVs primarily derive from apoptotic extracellular vesicles. nPMVs may provide an attractive source for developing EV-based pharmaceutical therapeutics., (© 2023. The Author(s).)

There is an increasing interest in cationic polymers as important constituents of non-viral gene delivery vectors. In the present study, we developed a versatile synthetic route for the production of covalent polymeric conjugates consisting of water-soluble depolymerized chitosan (dCS; M W 6-9 kDa) and low molecular weight polyethylenimine (PEI; 2.5 kDa linear, 1.8 kDa branched). dCS-PEI derivatives were evaluated based on their physicochemical properties, including purity, covalent bonding, solubility in aqueous media, ability for DNA condensation, and colloidal stability of the resulting polyplexes. They were complexed with non-integrating DNA vectors coding for reporter genes by simple admixing and assessed in vitro using liver-derived HuH-7 cells for their transfection efficiency and cytotoxicity. Using a rational screening cascade, a lead compound was selected (dCS-Suc-LPEI-14) displaying the best balance of biocompatibility, cytotoxicity, and transfection efficiency. Scale-up and in vivo evaluation in wild-type mice allowed for a direct comparison with a commercially available non-viral delivery vector (in vivo-jetPEI). Hepatic expression of the reporter gene luciferase resulted in liver-specific bioluminescence, upon intrabiliary infusion of the chitosan-based polyplexes, which exceeded the signal of the in vivo jetPEI reference formulation by a factor of 10. We conclude that the novel chitosan-derivative dCS-Suc-LPEI-14 shows promise and potential as an efficient polymeric conjugate for non-viral in vivo gene therapy.

Despite huge need in the medical domain and significant development efforts, artificial cells to date have limited composition and functionality. Although some artificial cells have proven successful for producing therapeutics or performing in vitro specific reactions, they have not been investigated in vivo to determine whether they preserve their architecture and functionality while avoiding toxicity. Here, these limitations are overcome and customizable cell mimic is achieved-molecular factories (MFs)-by supplementing giant plasma membrane vesicles derived from donor cells with nanometer-sized artificial organelles (AOs). MFs inherit the donor cell's natural cytoplasm and membrane, while the AOs house reactive components and provide cell-like architecture and functionality. It is demonstrated that reactions inside AOs take place in a close-to-nature environment due to the unprecedented level of complexity in the composition of the MFs. It is further demonstrated that in a zebrafish vertebrate animal model, these cell mimics show no apparent toxicity and retain their integrity and function. The unique advantages of highly varied composition, multicompartmentalized architecture, and preserved functionality in vivo open new biological avenues ranging from the study of biorelevant processes in robust cell-like environments to the production of specific bioactive compounds., Competing Interests: The authors declare no conflict of interest., (© 2020 The Authors. Published by WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim.)

Active targeting and specific drug delivery to parenchymal liver cells is a promising strategy to treat various liver disorders. Here, we modified synthetic lipid-based nanoparticles with targeting peptides derived from the hepatitis B virus large envelope protein (HBVpreS) to specifically target the sodium-taurocholate cotransporting polypeptide (NTCP; SLC10A1 ) on the sinusoidal membrane of hepatocytes. Physicochemical properties of targeted nanoparticles were optimized and NTCP-specific, ligand-dependent binding and internalization was confirmed in vitro. The pharmacokinetics and targeting capacity of selected lead formulations was investigated in vivo using the emerging zebrafish screening model. Liposomal nanoparticles modified with 0.25 mol% of a short myristoylated HBV derived peptide, that is Myr-HBVpreS2-31, showed an optimal balance between systemic circulation, avoidance of blood clearance, and targeting capacity. Pronounced liver enrichment, active NTCP-mediated targeting of hepatocytes and efficient cellular internalization were confirmed in mice by 111 In gamma scintigraphy and fluorescence microscopy demonstrating the potential use of our hepatotropic, ligand-modified nanoparticles., Competing Interests: DW, PU, SS, CK, TE, KS, PG, JB, YN, SS, JH, HM, GQ, WM, SU, JH No competing interests declared, (© 2019, Witzigmann et al.)

Injectable products are often the formulation of choice for new therapeutics; however, formulation in liquids often enhances degradation through hydrolysis. Thus, freeze-drying (lyophilization) is regularly used in pharmaceutical manufacture to reduce water activity. Here we examine its contribution to 'state of the art' and look at its future potential uses. A comprehensive search of patent databases was conducted to characterize the international patent landscape and trends in the use of freeze-drying. A total of 914 disclosures related to freeze-drying, lyophilization or drying of solid systems in pressures and temperatures equivalent to those of freeze-drying were considered over the period of 1992-2014. Current applications of sublimation technology were contrasted across two periods those with patents due to expire (1992-1993) and those currently filed. The number of freeze-drying technology patents has stabilized after initial activity across the biotechnology sector in 2011 and 2012. Alongside an increasing trend for patent submissions, freeze-drying submissions have slowed since 2002 and is indicative of a level of maturity.

We introduce active surfaces generated by immobilizing protein-polymer nanoreactors on a solid support for sensitive sugar alcohols detection. First, such selective nanoreactors were engineered in solution by simultaneous encapsulation of specific enzymes in copolymer polymersomes, and insertion of membrane proteins for selective conduct of sugar alcohols. Despite the artificial surroundings, and the thickness of the copolymer membrane, functionality of reconstituted Escherichia coli glycerol facilitator (GlpF) was preserved, and allowed selective diffusion of sugar alcohols to the inner cavity of the polymersome, where encapsulated ribitol dehydrogenase (RDH) enzymes served as biosensing entities. Ribitol, selected as a model sugar alcohol, was detected quantitatively by the RDH-nanoreactors with GlpF-mediated permeability in a concentration range of 1.5-9 mM. To obtain "active surfaces" for detecting sugar alcohols, the nanoreactors optimized in solution were then immobilized on a solid support: aldehyde groups exposed at the compartment external surface reacted via an aldehyde-amino reaction with glass surfaces chemically modified with amino groups. The nanoreactors preserved their architecture and activity after immobilization on the glass surface, and represent active biosensing surfaces for selective detection of sugar alcohols, with high sensitivity., (Copyright © 2016 Elsevier Ltd. All rights reserved.)