Facile assembly and loading of theranostic polymersomes via multi-impingement flash nanoprecipitation

Abstract

Flash nanoprecipitation (FNP) has proven to be a powerful tool for the rapid and scalable assembly of solid-core nanoparticles from block copolymers. The process can be performed using a simple confined impingement jets mixer and provides an efficient and reproducible method of loading micelles with hydrophobic drugs. To date, FNP has not been applied for the fabrication of complex or vesicular nanoarchitectures capable of encapsulating hydrophilic molecules or bioactive protein therapeutics. Here, we present FNP as a single customizable method for the assembly of bicontinuous nanospheres, filomicelles and vesicular, multilamellar and tubular polymersomes from poly(ethylene glycol)-bl-poly(propylene sulfide) block copolymers. Multiple impingements of polymersomes assembled via FNP were shown to decrease vesicle diameter and polydispersity, allowing gram-scale fabrication of monodisperse polymersomes within minutes. Furthermore, we demonstrate that FNP supports the simultaneous loading of both hydrophobic and hydrophilic molecules respectively into the polymersome membrane and aqueous lumen, and encapsulated enzymes were found to be released and remain active following vesicle lysis. As an example application, theranostic polymersomes were generated via FNP that were dual loaded with the immunosuppressant rapamycin and a fluorescent dye to link targeted immune cells with the elicited immunomodulation of T cells. By expanding the capabilities of FNP, we present a rapid, scalable and reproducible method of nanofabrication for a wide range of nanoarchitectures that are typically challenging to assemble and load with therapeutics for controlled delivery and theranostic strategies.

Keywords: Block copolymer; Drug delivery; Flash nanoprecipitation; Polymersome; Self-assembly.

Fig. 1

Overview of polymersome formation by flash nanoprecipitation (FNP). (A) A schematic of the CIJ mixer. (B) The structure of the diblock copolymer poly(ethylene glycol)-block-poly(propylene sulfide), and the weight fraction (f_PEG) dependent nanostructures known to form using the thin film hydration method. (C) A representative cryoTEM image of polymersomes formed by FNP, scale bar = 300 nm. Inset is a size distribution of polymersomes measured by nanoparticle tracking analysis (NTA), n = 6. Standard deviation is represented by the dotted lines.

Fig. 2

Fabrication of monodisperse polymersomes via flash nanoprecipitation. (A) DLS mean diameter of polymersomes formed after multiple impingements (1x–5x), or formed by thin film (TF) or solvent dispersion (SD) with (E) or without (NE) extrusion. Error bars are standard error, n = 5. (B) DLS size distribution of 5x impinged polymersomes the day of formation or after four days of storage at room temperature. Error bars are standard error, n = 3. (C–G) CryoTEM images of polymersomes formed after multiple impingements (1x–5x, respectively) with insets of DLS size distributions. X- and y- axes correspond to that of (B).

Fig. 3

Relationship between PEG weight fraction and morphology. (A) Diameter of nanostructures formed via FNP from PEG-bl-PPS copolymers of varying block lengths. Error bars represent the standard deviation of the nanostructure populations (PDI × Mean Diameter). Dotted area represents polymersome-forming samples. Arrows point out samples of note. †Sample formed using DMF as the organic solvent, rather than THF. ‡Sample formed using water instead of 1xPBS. (B–G) Weight fractions of PEG responsible for forming specific nanostructures via flash nanoprecipitation, paired with cartoon and representative cryoTEM images. All scale bars = 100 nm, with the exception of scale bars within (B) and (E), which are 300 nm. Sample number is listed in the upper corner of each cryoTEM image, and the number of impingements used is listed for each morphology. See Table 1 for details of copolymers and Fig. S5 for low magnification images.

Fig. 4

Loading of polymersomes with small molecules and macromolecules. (A) Loading efficiency of small molecules and macromolecules. (B) Live-cell confocal microscopy image of polymersome uptake and delivery of GFP in a bone marrow-derived dendritic cell. Polymersomes were loaded with the hydrophobic ethyl eosin (red) and hydrophilic GFP (green). Cells were additionally stained with SYTO 61 (yellow) and lysotracker (blue). Scale bar = 5 microns. (C) Graphical representation of experimental setup. Alkaline phosphatase (AP) is represented by circles, BCIP by triangles, and NBT by squares. The product of the enzymatic reaction, formazan, absorbs strongly at 620 nm and is represented by a star. (D) Time-course of enzyme activity assay. Y-axis represents fold increase over original absorbance reading. Error bars represent standard deviation, n = 4. Statistical significance determined by 2-way ANOVA, * p<0.05 and *** p<0.001.

Fig. 5

In vivo delivery of theranostic rapamycin/DiD-loaded polymersomes formed by flash nanoprecipitation. (A) Percentage of CD8+ T cells (CD45+ CD3+ CD4− CD8+) and CD4+ T cells (CD45+ CD3+ CD4+ CD8−) within the total T cell population (CD45+ CD3+) and percentage of CD8+ DCs (CD11c+ I-A/I-E+ CD8+) within the total DC (CD11c+) population. Treatment groups were rapamycin polymersomes (R-PS), free rapamycin, blank polymersomes, and vehicle (PBS). (B) T cell subpopulations as a percent of total T cell population for all four treatment groups. (C) T cells in the spleen and lymph nodes, as a percentage of CD45+ cells. (D) Median fluorescence intensity of the polymersome channel for selected cell populations in the spleen and lymph nodes of mice administered rapamycin/DiD-loaded polymersomes. N=3, statistical significance determined by Tukey’s multiple comparisons test, * p < 0.05, ** p < 0.01, ***p < 0.001, ****p < 0.0001.