The effect of low temperature on poly(3-methyl-N-vinylcaprolactam)-b-poly(N-vinylpyrrolidone) diblock copolymer nanovesicles assembled from all-aqueous media

Nanosized polymeric vesicles (polymersomes) self-assembled from double hydrophilic copolymers of poly(3-methyl-N-vinylcaprolactam)_n-b-poly(N-vinylpyrrolidone)_m (PMVC_n-b-PVPON_m) using all aqueous media are a promising platform for biomedical applications, because of their superior stability over liposomes in vivo and high loading capacity. Herein, we explored the temperature-sensitive behavior of PMVC₅₈-b-PVPON₆₅ vesicles using transmission electron microscopy (TEM), dynamic light scattering (DLS), atomic force microscopy (AFM), and small-angle neutron scattering (SANS) in response to lowering the solution temperature from 37 to 25, 20, 14 and 4 °C. The copolymer vesicles with an average size of 350 nm at 37 °C were assembled from the diblock copolymer dissolved in aqueous solution at 4 °C. We show that while the polymersome's size gradually decreases upon the temperature decrease from 37 to 4 °C, the average shell thickness increases from 17 nm to 25 nm, respectively. SANS study revealed that the PMVC₅₈-b-PVPON₆₅ vesicle undergoes a gradual structure evolution from a dense-shell vesicle at 37-25 °C to a highly-hydrated shell vesicle at 20-14 °C to molecular chain aggregates at 4 °C. From SANS contrast matching study, this vesicle behavior is found to be driven by the gradual rehydration of PMVC block at 37-14 °C. The shell hydration at 20-14 °C also correlated with the 4.4-fold decrease in the relative fluorescence intensity from vesicle-encapsulated fluorescent dye, indicating ∼80% of the dye release within 12 hours after the vesicle exposure to 14 °C. No significant (<5%) dye release was observed for the vesicle solutions at 37-20 °C, indicating excellent cargo retention inside the vesicles. Our study provides new fundamental insights on temperature-sensitive polymer vesicles and demonstrates that the copolymer assembly into polymersomes can be achieved by decreasing a copolymer aqueous solution temperature below 14 °C followed by solution exposure to ≥20 °C. This type of all-aqueous assembly, instead of nanoprecipitation from organic solvents or solvent exchange, can be highly desirable for encapsulating a wide range of biological molecules, including proteins, peptides, and nucleic acids, into stable polymer vesicles without a need for organic solvents for dissolution of the copolymers that are amphiphilic at physiologically relevant temperatures of 20-37 °C.