Sequential intracellular release of water-soluble cargos from Shell-crosslinked polymersomes

Abstract

Polymer vesicles, i.e. polymersomes (PS), present unique nanostructures with an interior aqueous core that can encapsulate multiple independent cargos concurrently. However, the sequential release of such co-loaded actives remains a challenge. Here, we report the rational design and synthesis of oxidation-responsive shell-crosslinked PS with capability for the controlled, sequential release of encapsulated hydrophilic molecules and hydrogels. Amphiphilic brush block copolymers poly(oligo(ethylene glycol) methyl ether methacrylate)-b-poly(oligo(propylene sulfide) methacrylate) (POEGMA-POPSMA) were prepared to fabricate PS via self-assembly in aqueous solution. As a type of unique drug delivery vehicle, the interior of the PS was co-loaded with hydrophilic molecules and water-soluble poly(N-isopropylacrylamide) (PNIPAM) conjugates. Due to the thermosensitivity of PNIPAM, PNIPAM conjugates within the PS aqueous interior underwent a phase transition to form hydrogels in situ when the temperature was raised above the lower critical solution temperature (LCST) of PNIPAM. Via control of the overall shell permeability by oxidation, we realized the sequential release of two water-soluble payloads based on the assumption that hydrogels have much smaller membrane permeability than that of molecular cargos. The ability to control the timing of release of molecular dyes and PNIPAM-based hydrogels was also observed within live cells. Furthermore, leakage of hydrogels from the PS was effectively alleviated in comparison to molecular cargos, which would facilitate intracellular accumulation and prolonged retention of hydrogels within the cell cytoplasm. Thus, we demonstrate that the integration of responsive hydrogels into PS with crosslinkable membranes provides a facile and versatile technique to control the stability and release of water-soluble cargos for drug delivery purposes.

Keywords: Oxidation-responsive; Polymersomes; Self-assembly; Sequential release; Shell crosslinking.

Figure 1

POEGMA-POPSMA block copolymers (BCP) self-assemble into polymersomes (PS) and support the size-dependent dual release of hydrophilic payloads. (a) Chemical structures of oxidation-responsive amphiphilic POEGMA-POPSMA BCP used for the fabrication of PS with crosslinkable membranes, and (b) schematic of sequential release from the shell crosslinked PS at 37 °C under a stimulus of oxidation.

Figure 2

CryoTEM images of nanostructures fabricated by the self-assembly of POEGMA-POPS₁₇MA₃ (a) and POEGMA-POPS₇MA₈ (b–f) in PBS. (a) Coexistence of large size-distributed PS and smaller micelles in a POEGMA-POPS₁₇MA₃ dispersion, (b) Large size distributions were observed for uncrosslinked POEGMA-POPS₇MA₈ PS prior to extrusion, (c) monodisperse single-layer uncrosslinked PS obtained following extrusion, (d) shell crosslinked PS, (e) PNIPAM-RhB encapsulated shell crosslinked PS (cryo-fixed at 37 °C), and (f) PNIPAM-RhB encapsulated uncrosslinked PS (cryo-fixed at 37 °C displayed frequent vesicle rupture (red arrows) and PNIPAM-RhB hydrogels. Scale bar = 200 nm.

Figure 3

Physicochemical characteristics of C-PS in PBS at 25 °C by DLS (a) and SAXS (b).

Figure 4

Crosslinking of PS membranes increases stability and support in situ gelation of PNIPAM for retention of large encapsulated cargo. (a) Time-dependent evolution of normalized absorbance (turbidity) for PS with different degrees of crosslinking (0%, 17%, 32%, 44%, and 52%) in PBS with 1.63 M of H₂O₂ (5%). (b) Leakage profiles of calcein from U-PS and C-PS at 25 °C. (c) Intensity-average hydrodynamic diameter distributions recorded for U-PS and C-PS loaded with PNIPAM-RhB in PBS after incubation at 37 °C for 24 h. (d) Leakage profiles of calcein and PNIPAM-RhB hydrogels co-loaded C-PS in PBS at 37 °C. Data represent means ± standard deviations (n = 3). Statistical significance: *p ≤ 0.01, **p ≤ 0.005, ***p ≤ 0.0001.

Figure 5

Crosslinked PS display reduced release rates for encapsulated PNIPAM hydrogels in comparison to small hydrophilic payloads. (a) Oxidation-triggered release of calcein at 37 °C from C-PS upon incubation with varying H₂O₂ concentrations (0, 0.1, 100, and 163 mM in PBS). (b) In vitro co-release profiles of calcein and PNIPAM-RhB at 37 °C from C-PS in the presence of H₂O₂ (163 mM, 0.5% in PBS). Data represent means ± standard deviations (n = 3). Statistical significance: *p ≤ 0.01, **p ≤ 0.005, ***p ≤ 0.0001.

Figure 6

Confocal microscopy images of calcein (green channel) and PNIPAM-RhB (red channel) co-loaded C-PS upon incubation in PBS with varying H₂O₂ concentrations at 37 °C for 24 h. From left to right: (a) PBS, (b) 163 mM (0.5%) H₂O₂, (c) 326 mM (1.0%) H₂O₂, (c) 652 mM (2.0%) H₂O₂. Scale bar = 5 µm.

Figure 7

Confocal fluorescence images of A2780 cells. (a) Cells were incubated with Nile blue chloride (red channel) and PNIPAM-Rh B (green channel) co-loaded C-PS for 1 h and 24 h. Nucleus were stained by DAPI. (b) Cells were first treated with PNIPAM-RhB loaded C-PS for 1 h, and then incubated with fresh medium for 1 h and 24 h. Nucleus and lysosome were stained by Hoechst 33342 and LysoTracker Green, respectively. Scale bar = 10 µm.

Scheme 1

Synthetic routes employed for the preparation of OPSMA monomers and POEGMA-POPSMA comb-shaped amphiphilic BCP.