The Role of Membrane Fluidization in the Gel-Assisted Formation of Giant Polymersomes

<div><p>Polymersomes are being widely explored as synthetic analogs of lipid vesicles based on their enhanced stability and potential uses in a wide variety of applications in (e.g., drug delivery, cell analogs, etc.). Controlled formation of giant polymersomes for use in membrane studies and cell mimetic systems, however, is currently limited by low-yield production methodologies. Here, we describe for the first time, how the size distribution of giant poly(ethylene glycol)-poly(butadiene) (PEO-PBD) polymersomes formed by gel-assisted rehydration may be controlled based on membrane fluidization. We first show that the average diameter and size distribution of PEO-PBD polymersomes may be readily increased by increasing the temperature of the rehydration solution. Further, we describe a correlative relationship between polymersome size and membrane fluidization through the addition of sucrose during rehydration, enabling the formation of PEO-PBD polymersomes with a range of diameters, including giant-sized vesicles (>100 μm). This correlative relationship suggests that sucrose may function as a small molecule fluidizer during rehydration, enhancing polymer diffusivity during formation and increasing polymersome size. Overall the ability to easily regulate the size of PEO-PBD polymersomes based on membrane fluidity, either through temperature or fluidizers, has broadly applicability in areas including targeted therapeutic delivery and synthetic biology.</p></div

Summary of the different polymers tested, their abbreviations used in the text and the molecular weight (M_w).

<p>Summary of the different polymers tested, their abbreviations used in the text and the molecular weight (M_w).</p

Dependency of vesicle size on different rehydration temperatures.

<p>PEO-PBD polymersomes were generated in water on 1% agarose gels for 30 min at varying temperatures on a hot plate. (a) Epifluorescence photomicrographs (scale bar = 10 μm), (b) average diameters (± standard error of the mean), and (c) frequency distribution plots for polymersomes formed at different temperatures.</p

Diffusion coefficients (mean ± standard error) estimated from FRAP data for polymersomes formed from different polymers.

<p>Diffusion coefficients (mean ± standard error) estimated from FRAP data for polymersomes formed from different polymers.</p

Polymersomes were formed in different buffers and from a variety of polymers following rehydrated with water at 40°C for 1 h on an agarose gel.

<p>(a) Epifluorescence photomicrographs of PEO-PBD polymersomes formed using various different rehydration solutions, or (b) with different polymer compositions (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158729#pone.0158729.t001" target="_blank">Table 1</a> and Table A in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158729#pone.0158729.s001" target="_blank">S1 File</a> for more details on the different polymers). Scale bars = 10 μm.</p

Effects of membrane fluidization on the formation of polymersomes.

<p>(a) Epifluorescence photomicrographs of PEO-PBD polymersomes formed on either agarose gels in water (left two images) or agarose gels in sucrose (right two images) and rehydrated in either water or sucrose as indicated at the top of the image. Scale bar = 10 μm. (b) Correlation of polymersome size (measured for over 80 polymersomes) and diffusion coefficients (calculated for at least five different polymersomes). X-error bars are standard error of the mean for the polymersome diameters. Y-error bars are standard error of the mean for the diffusion coefficients.</p

Fluorescence recovery after photobleaching (FRAP) analysis shows that polymersome membranes are fluid.

<p>(a) Epifluorescence imaging of a representative PEO-PBD polymersome pre-, during and post-fluorescence bleaching. The region of the membrane that was bleached is circled. Scale bar = 10 μm. (b) Time-dependent fluorescence recovery profiles for different polymers.</p

Comparison of polymersomes formed by electroformation and gel-assisted rehydration.

<p>(a) Representative epifluorescence photomicrographs depicting PEO-PBD, positively-charged PEO-PBD (NH³⁺) and negatively-charged PEO-PBD (COO^-) polymersomes formation using platinum wire electroformation. (b) Epifluorescence photomicrographs depicting polymersome formation using gel-assisted rehydration after 1 h on an agarose gel at 40°C. Scale bar = 10 μm.</p