The integration of therapeutic biomolecules, such as proteins and peptides, in nanovesicles is a widely used strategy to improve their stability and efficacy. However, the translation of these promising nanotherapeutics to clinical tests is still challenged by the complexity involved in the preparation of functional nanovesicles and their reproducibility, scalability, and cost production. Here we introduce a simple one-step methodology based on the use of CO2-expanded solvents to prepare multifunctional nanovesicle-bioactive conjugates. We demonstrate high vesicle-to-vesicle homogeneity in terms of size and lamellarity, batch-to-batch consistency, and reproducibility upon scaling-up. Importantly, the procedure is readily amenable to the integration/encapsulation of multiple components into the nanovesicles in a single step and yields sufficient quantities for clinical research. The simplicity, reproducibility, and scalability render this one-step fabrication process ideal for the rapid and low-cost translation of nanomedicine candidates from the bench to the clinic.

Nanopharmaceutics composed of a carrier and a protein have the potential to improve the activity of therapeutical proteins. Therapy for lysosomal diseases is limited by the lack of effective protein delivery systems that allow the controlled release of specific proteins to the lysosomes. Here we address this problem by developing functional polyelectrolyte-based nanoparticles able to promote acidic pH-triggered release of the loaded protein. Trimethyl chitosan (TMC) was synthesized and allowed to form polyelectrolyte complexes (PECs) with the lysosomal enzyme α-GAL through self-assembly and ionotropic gelation, with average particle size <200 nm, polydispersity index (PDI) <0.2, ζ potential of ∼ 20 mV, and a protein loading efficiency close to 65%. These polyelectrolyte nanoparticles were stable and active under physiological conditions and able to release the enzyme at acidic pH, as demonstrated by in situ atomic force microscopy (AFM). These nanoparticles were further functionalized with Atto 647N for single-particle characterization and tracking their cellular uptake and fate using high-resolution fluorescence microscopy. In contrast with their precursor, TMC, PECs were efficiently internalized by human endothelial cells and mostly accumulated in lysosomal compartments. The superior physicochemical characteristics of the TMC/α-GAL PECs together with their excellent cellular uptake properties indicate their enormous potential as advanced protein delivery systems for the treatment of lysosomal storage diseases.

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Proton-translocating ATPases are central to biological energy conversion. Although eukaryotes contain specialized F-ATPases for ATP synthesis and V-ATPases for proton pumping, eubacteria and archaea typically contain only one enzyme for both tasks. Although many eubacteria contain ATPases of the F-type, some eubacteria and all known archaea contain ATPases of the A-type. A-ATPases are closely related to V-ATPases but simpler in design. Although the nucleotidebinding and transmembrane rotor subunits share sequence homology between A-, V-, and F-ATPases, the peripheral stalk is strikingly different in sequence, composition, and stoichiometry. We have analyzed the peripheral stalk of Thermus thermophilus A-ATPase by using phage display-derived single-domain antibody fragments in combination with electron microscopy and tandem mass spectrometry. Our data provide the first direct evidence for the existence of two peripheral stalks in the A-ATPase, each one composed of heterodimers of subunits E and G arranged symmetrically around the soluble A 1 domain. To our knowledge, this is the first description of phage display-derived antibody selection against a multi-subunit membrane protein used for purification and single particle analysis by electron microscopy. It is also the first instance of the derivation of subunit stoichiometry by tandem mass spectrometry to an intact membrane protein complex. Both approaches could be applicable to the structural analysis of other membrane protein complexes.F-, V-, and A-ATPases 2 are evolutionary related protein complexes (1) that couple ATP synthesis/hydrolysis with proton (or Na ϩ ) translocation across membranes (2-4). A-and V-ATPases are evolutionarily closer to each other than they are to F-ATPases (5). However, A-ATPases are functionally more similar to F-ATPases because both synthesize ATP using energy derived from proton translocation (5). V-ATPases work in reverse by actively pumping protons through membranes using ATP hydrolysis as the driving force (6).Although eukaryotes contain both types of ATPases, each one highly specialized in its physiological function, archaea and eubacteria typically contain only one. Both eubacterial F-ATPases and eubacterial and archaeal V-ATPases are simpler than their eukaryotic counterparts but are functionally more versatile in that they can operate in both directions. Archaeal and eubacterial V-ATPases are closely related and are often referred to as A-ATPases (4).F-, V-, and A-ATPases share an overall conservation of structure that includes a water-soluble F 1 /V 1 /A 1 domain and a membrane-bound F o /V o /A o domain (7-10). The Thermus thermophilus ATPase-active A 1 domain is composed of a head group that contains a heterotrimer of the nucleotide-binding proteins A and B and a central stalk composed of proteins C, D, and F (11). The proton-translocating A o domain contains a ring of L proteolipids and a single copy of protein I that is located adjacent to the ring. The L ring, in association with the central stalk components (CDF), for...