Aquaporin-Based Biomimetic Polymeric Membranes: Approaches and Challenges

Abstract

In recent years, aquaporin biomimetic membranes (ABMs) for water separation have gained considerable interest. Although the first ABMs are commercially available, there are still many challenges associated with further ABM development. Here, we discuss the interplay of the main components of ABMs: aquaporin proteins (AQPs), block copolymers for AQP reconstitution, and polymer-based supporting structures. First, we briefly cover challenges and review recent developments in understanding the interplay between AQP and block copolymers. Second, we review some experimental characterization methods for investigating AQP incorporation including freeze-fracture transmission electron microscopy, fluorescence correlation spectroscopy, stopped-flow light scattering, and small-angle X-ray scattering. Third, we focus on recent efforts in embedding reconstituted AQPs in membrane designs that are based on conventional thin film interfacial polymerization techniques. Finally, we describe some new developments in interfacial polymerization using polyhedral oligomeric silsesquioxane cages for increasing the physical and chemical durability of thin film composite membranes.

Keywords: aquaporins; biomimetic membranes; block copolymers; membrane proteins; microfluidics; polyamide layer; polyhedral oligomeric silsesquioxanes; protein-polymer-interactions; proteopolymersomes.

Figure 1

Schematic drawing of aggregate morphologies as a function of mPAR. PB₁₂-PEO₁₀ undergoes four transitions. Surprisingly, the vesicle shape remained at significantly higher densities at block copolymers, compared to a standard lipid like 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). The mPAR of the one-molecule-bilayer-forming ABA triblock copolymers was divided by two enabling direct comparison with the PB-PEO diblock copolymers and DOPE, both forming bilayers. The morphologies in full color are the main morphologies, pale colors denote coexisting morphologies. Adapted from [34].

Figure 2

TEM images of aggregate morphologies as a function mPAR. Where the PMOXA-PDMS-PMOXA copolymers self-assemble to vesicles, PB-PEO forms network- and sperm-like structures and only after incorporation of AQP0 vesicular structures are observed. Scale bar is 200nm. Adapted from [33].

Figure 3

Overview of relevant parameters for membrane protein incorporation into amphiphilic block copolymers.

Figure 4

Normalized light scattering vs. time for proteo- and polymersomes of PB₄₅-PEO₁₄ and PB₃₃-PEO₁₈ at an mPAR of 1:100. For PB₄₅-PEO₁₄ the apparent water permeability is slightly decreased for the proteopolymersomes versus polymersomes, whereas for PB₃₃-PEO₁₈ it is slightly increased.

Figure 5

FF-TEM images of PB₄₅-PEO₁₄ proteo—(b,c,e,f) and polymersomes (a,d). All vesicles revealed spots, potentially not from AqpZ but rather collapsed PB chains (a–c) or bad fracturing artifacts (d–f). Scale bar is 100 nm.

Figure 6

(a) Correlation diagram of proteopolymersomes and AQP10-GFP stock solution as a function of correlation time τ against autocorrelation function G(τ). The higher autocorrelation signal indicates a lower number of particles in the confocal volume, due to slower diffusion time. (b) Fluorescence lifetimes of the same samples as a function of lifetime against intensity signal. Where the intensities varied, the fluorescence lifetime was in a comparable range.

Figure 7

SAXS data for proteo- and polymersomes of PB₄₅-PEO₁₄ (a) and PB₃₃-PEO₁₈ (b). The fits were obtained using a vesicle model consisting of three concentric spherical shells. To fit the polymersomes of PB₃₃-PEO₁₈, it was necessary to include an additional contribution from block-copolymer micelles as shown in the insert.

Figure 8

Schematic overview of all published designs for ABPMs and ABLMs. Pioneer work is mainly done by Kumar and Aquaporin AIS. The most experimental designs has been done by NUS, where NTU published the most promising layer embedment ABLMs. The main recent work is on LbL-based electrostatic binding, for example binding of proteoliposomes on a polyelectrolyte layer [142].

Figure 9

Chemical structure of POSS and TMC and the resulting AL. POSS as the amine linker generate a highly stable and well-defined AL with TMC.

Figure 10

FTIR diagram of POSS/polymersomes+TMC AL (labelled red) and POSS+TMC control AL (labelled blue) as a function of wavelength against absorption. The AL with polymersomes had an absorption peak around 3000 cm⁻¹, that responds to PB and PEO, indicating their presence in the AL, where the characteristic absorption peaks for PA bonds and POSS were present as well.

Figure 11

SEM images of POSS+TMC AL (a,b) and of POSS/polymersomes+TMC AL (c–e) with schematic sketches, which part of the layer is being captured. Images were taken from different parts of the flakes (labelled green in the sketch) of the AL, that were generated during the SEM preparation. The AL without polymersomes was smooth and well-defined, which remained on the organic side when polymersomes were added. The aqueous side was covered with loosely attached and half-covered polymersomes (dotted circle in (d)). A few could be observed inside the AL (arrows in (d)). Scale bar is 3 µm.

Figure 12

(a) Schematic sketch of the microfluidic chamber and micrographs of POSS/proteopolymersomes+TMC AL and (b) micrograph of the compartment. The aqueous phase reached into the other compartment. After introducing the organic phase, a well-defined AL formed. Scale bar is 50 µm.

Figure 13

FTIR analysis of supported POSS/polymersomes+TMC AL (red) and POSS+TMC control AL (blue) on MF PES and pure MF PES (black). The PES supporting material had high absorption and interfered with many absorption peaks. A subtraction from the absorption spectra of pure PES resulted in negative peaks. We therefore only normalized the spectra. PB-PEO was present in the AL with polymersomes; however, the PA formation was strongly suppressed.

Figure 14

SEM images of MF PES (a,b) supported POSS+TMC AL on MF PES (c) and supported POSS/polymersomes+TMC on MF PES (d,e). Schematic sketch of polymersome coverage left to (e). Micropores of the MF PES were covered completely by the POSS+TMC AL. After addition of polymersomes, small bumps with dimensions similar to the polymersomes were observed on the organic faced side of the AL. Greater bumps may be attributed to accumulations of covered polymersomes. Scale bar is 3 µm.