Quantification of intracellular payload release from polymersome nanoparticles

Abstract

Polymersome nanoparticles (PMs) are attractive candidates for spatio-temporal controlled delivery of therapeutic agents. Although many studies have addressed cellular uptake of solid nanoparticles, there is very little data available on intracellular release of molecules encapsulated in membranous carriers, such as polymersomes. Here, we addressed this by developing a quantitative assay based on the hydrophilic dye, fluorescein. Fluorescein was encapsulated stably in PMs of mean diameter 85 nm, with minimal leakage after sustained dialysis. No fluorescence was detectable from fluorescein PMs, indicating quenching. Following incubation of L929 cells with fluorescein PMs, there was a gradual increase in intracellular fluorescence, indicating PM disruption and cytosolic release of fluorescein. By combining absorbance measurements with flow cytometry, we quantified the real-time intracellular release of a fluorescein at a single-cell resolution. We found that 173 ± 38 polymersomes released their payload per cell, with significant heterogeneity in uptake, despite controlled synchronisation of cell cycle. This novel method for quantification of the release of compounds from nanoparticles provides fundamental information on cellular uptake of nanoparticle-encapsulated compounds. It also illustrates the stochastic nature of population distribution in homogeneous cell populations, a factor that must be taken into account in clinical use of this technology.

Challenges for the Self-Assembly of Poly(Ethylene Glycol)–Poly(Lactic Acid) (PEG-PLA) into Polymersomes: Beyond the Theoretical Paradigms

“…One should keep in mind that DLS is more adequate than NTA for the determination of nanostructures hydrodynamic diameter. Nonetheless, NTA allows an estimation of the concentration of PL [ 21 ] and our results clearly show that the PEG 45 -PLA 69 system after the extrusion resulted in more diluted systems in terms of number of polymersomes due to the initial loss of polymeric material in the bulk fraction after the film hydration, what was already visually observed. The PLs prepared with higher concentration of copolymer (0.1% ( m / v )) ( Figure 8 ) resulted in narrower size distribution and PDI values of 0.345, 0.144 and 0.081 for PEG 45 -PLA 69 , PEG 114 -PLA 153 and PEG 114 -PLA 180, respectively.…”

Nanocarrier systems assembled from PEGylated hyperbranched poly(arylene oxindole)

“…We believe that these larger particles resulted from the lateral fusion of NPs during the longer time allowed for self-assembly, as reported elsewhere [1,5,6,56]. According to the literature, these particles would be classified as hyperbranched polymer vesicles composed of a bilayer membrane and hydrophilic interior, which have previously shown their potential as DDSs [5,57], but more studies are required to confirm this hypothesis.…”

Probing and Tuning the Permeability of Polymersomes

“… 25 In a similar study, the self-quenching dye fluorescein was encapsulated within poly(ethylene glycol)- b -poly(ε-caprolactone) (PEG- b -PCL) polymersomes above the quenching concentration. 26 Upon permeation through the polymersome membrane into the exterior solution, the dye would fluoresce, as observed by spectrophotometry, allowing quantification of the polymersome membrane permeability. This method has also been applied to the release of other types of encapsulated cargo, such as the drug doxorubicin from PS- b -PAA polymersomes.…”