Avidin Localizations in pH-Responsive Polymersomes for Probing the Docking of Biotinylated (Macro)molecules in the Membrane and Lumen

Abstract

To mimic organelles and cells and to construct next generation therapeutics, asymmetric functionalization and location of proteins for artificial vesicles is thoroughly needed to emphasize the complex interplay of biological units and systems through spatially separated and spatiotemporal controlled actions, release and communications. For the challenge of vesicle (= polymersome) construction, the membrane permeability and the location of the cargo are important key characteristics that determine the potential applications of them. Herein, an in situ and post loading process of avidin in pH-responsive and photocrosslinked polymersomes is developed and characterized. Firstly, loading efficiency, main location (inside, lumen, outside) and release of avidin under different conditions have been validated, including the pH-stable presence of avidin in polymersomes´ membrane outside and inside. This advantageous approach allows to selectively functionalize the outer and inner membrane as well the lumen with several bio(macro)molecules, generally suited for the construction of asymmetrically functionalized artificial organelles. In addition, a FRET effect was used to study the permeability or uptake of polymersomes membrane against a broad range of biotinylated (macro)molecules (different typology, sizes and shapes) at different conditions.

Multivalent Protein‐Loaded pH‐Stable Polymersomes: First Step toward Protein Targeted Therapeutics

“…[ 43,44,46 ] Further studies extended these results to the post loading approach. [ 65,66 ] For both loading approaches, the same amount of proteins was used (1 mg BCP per mL + 0.1 mg mL −1 protein). Successful loading by the Psomes was proven by fluorescence spectroscopy using labeled proteins (HSA‐FITC or Avidin/bFITC, Supporting Information).…”

“…Using in situ loading, the protein will be mainly found inside and anchored in the membrane. [ 46,66 ] Using post loading, the membrane and surface of Psomes will be more favored. [ 50,65 ] Thus, the choice of loading approach will depend on the subsequent application of those protein‐loaded Psomes desired.…”

Artificial Organelles with Orthogonal‐Responsive Membranes for Protocell Systems: Probing the Intrinsic and Sequential Docking and Diffusion of Cargo into Two Coexisting Avidin–Polymersomes

“…[ 68 ] In line with this, Avidin‐Psome A possesses the same key characteristics for pH* and pH 0 as found for pure Psome A validated under the same conditions. [ 75 ] Moreover, Avidin‐Psome A demonstrates a high pH stability and does not release avidin biomacromolecules at neutral and acidic pH. This allowed us to study successfully the diffusion pathways and docking processes of biotinylated cargo on collapsed and swollen Avidin‐Psome A, but also to identify the location(s) of docked cargo.…”

Increased Protein Encapsulation in Polymersomes with Hydrophobic Membrane Anchoring Peptides in a Scalable Process

“…Many polymersome formulations combine polymersomes with proteins, in particular by encapsulation of enzymes within the polymersome lumen. However, the encapsulation efficiency largely depends on the method of polymersome formation, the chemical properties of the used copolymers and even on the properties of the proteins themselves, leading to largely varying degrees of encapsulation, with reported values ranging between <1% and 100% [8,[17][18][19][20]. The formation of polymersomes in STRs is done by injecting the polymer in organic solvent into the aqueous phase.…”