Artificial Organelles with Orthogonal‐Responsive Membranes for Protocell Systems: Probing the Intrinsic and Sequential Docking and Diffusion of Cargo into Two Coexisting Avidin–Polymersomes

Abstract The challenge of effective integration and use of artificial organelles with orthogonal‐responsive membranes and their communication in eukaryotic protocells is to understand the intrinsic membrane characteristics. Here, a novel photo‐crosslinked and pH‐responsive polymersome (Psome B) with 2‐(N,N′‐diisopropylamino)ethyl units in the membrane and its respective Avidin‐Psome B hybrids, are reported as good candidates for artificial organelles. Biotinylated (macro)molecules are able to dock and diffuse into Avidin‐Psome B to carry out biological activity in a pH‐ and size‐dependent manner. Combined with another polymersome (Psome A) with 2‐(N,N′‐diethylamino)ethyl units in the membrane, two different pH‐responsive polymersomes for mimicking different organelles in one protocell system are reported. The different intrinsic docking and diffusion processes of cargo (macro)molecules through the membranes of coexisting Psome A and B are pH‐dependent as confirmed using pH titration–dynamic light scattering (DLS). Psome A and B show separated “open”, “closing/opening”, and “closed” states at various pH ranges with different membrane permeability. The results pave the way for the construction of multicompartmentalized protocells with controlled communications between different artificial organelles.


Gel Permeation Chromatography (GPC).
The weight average molecular weight (Mw), number average molecular weight (Mn) and molar mass distributions (Ð) of block copolymers were measured using SEC equipped with a MALLS detector (MiniDAWN-LS detector, Wyatt Technology, USA) and a viscosity/refractive index (RI) detector (ETA-2020, WGE Dr. Bures, Germany). The column (PL MIXED-C with a pore size of 5 μm, 300x7.5 mm) and the pump (HPLC pump, Agilent 1200 series) were from Agilent Technologies (USA). THF was used as an eluent (stabilized with 0.025 % BHT) with a flow rate of 1 mL/min. The calibration was based on polystyrene standards ranging from 1300 to 377400 g/mol.
Hollow Fiber Filtration (HFF). HFF was carried out using KrosFlo Research Iii System. This device was equipped with a separation module made of polyether sulfone membrane (MWCO: 500 kDa, SpectrumLabs, USA). The transmembrane pressure was from 70 mbar to 150 mbar with a flow rate of 15 mL/min. All the samples were purified by washing continuously with 1 mM PBS buffer at pH from 8.0 to 5.0.

Dynamic Light Scattering (DLS). DLS measurements of aqueous polymersome solutions
(0.2~1 mg/mL) were performed using a Zetasizer Nano-series instrument (Malvern Instruments, UK) equipped with Dispersion Technology Software (version 5.00). The measurements were carried over a range of pH at 25°C. The data was collected using the NIBS (non-invasive back-scatter) method using a Helium-Neon laser (4 mW, l = 632.8 nm) and a fixed angle of 173°.
Zeta-potential measurements. Zeta potential of the polymersome solution (0.5 mg/mL) was determined by Zetasizer Nano-series instrument (Malvern Instruments, UK) through electrophoretic light scattering.

UV lamp for crosslinking of polymersome. EXFO Omnicure 1000 (Lumen Dynamics
GroupInc., Canada) equipped with a high-pressure mercury lamp as UV source was used for crosslinking the polymersomes. The crosslinking process were performed with 1~1.5 mL of polymersome solution for from 90 to 180 seconds.
UV-Vis Spectroscopy. UV-vis analysis was performed using Specord 210 Plus double beam UV-vis spectrophotometer (analytikjena, Germany). Samples were measured at desired wavelength range in semi-micro cuvettes (Brand GmbH).

Cryogenic transmission electron microscopy (cryo-TEM).
Cryo-TEM images were acquired using Libra 120 microscope (Carl Zeiss Microscopy GmbH, Oberkochen, Germany) at an acceleration voltage of 120 kV. Samples were prepared by dropping 2 µL of polymersome solution on copper grids coated with holey carbon foil (so-called Lacey type). A piece of filter paper was used to remove the excess water; the sample was then rapidly frozen in liquid ethane at -178 °C. The blotting with the filter paper and plunging into liquid ethane was done in a Leica GP device (Leica Microsystems GmbH, Wetzlar, Germany). All images were recorded in bright field at -172 °C. The diameter and membrane thickness of the polymersome were determined from cryo-TEM images by using ImageJ Sofware. The average of polymersome diameter was calculated by analyzing more than 100 particles. The average of membrane thickness was calculated by analyzing 30 particles.
Asymmetrical flow field flow fractionation (AF4). AF4 measurements were performed with an Eclipse DUALTEC system (Wyatt Technology Europe, Germany) with 1 mM PBS buffer at pH 6.5, containing 200 mg/L NaN3 to prevent bacteria or algae contamination as carrier liquid.
The channel spacer, made of poly(tetrafluoroethylene), had a thickness of 490 µm. The dimensions of the channel were 26.5 cm in length and from 2.1 to 0.6 cm in width. The membranes used as accumulation wall were composed of polyethersulfone with a molecular weight cut off (MWCO) of 10 kDa (Nadir, Germany). Flow rates were controlled with an Agilent Technologies 1260er series isocratic pump equipped with vacuum degasser. The Technologies Deutschland GmbH). The data collection and calculation of molecular weights and radii were performed by Astra 7.3.219 software (Wyatt Technologies, USA). Cross flow rate (Fx) profile was optimized to achieve optimal fractionation of free molecules from Psomes within the same elution ( Figure S1). The following protocol was applied: detector flow was set to 0.5 mL/min, focusing was performed with focus flow (Ff) 2.5 mL/min for 4 min followed by an isocratic elution step with a Fx of 2 mL/min for 5 min followed by an exponential Fx gradient from 2 to 0 mL/min within 20 min. The last step proceeds without Fx (0 mL/min) for PEG45-Br macroinitiator ((107 mg, 0.05 mmol) (3), 2,2′-bipyridine (14.5 mg, 0.093 mmol), DEAEMA (604 mg, 3.257 mmol) (9), and photo-crosslinker A (247 mg, 0.931 mmol) (8) were mixed in a Schlenk tube with a stirring bar. The compounds were dissolved in 2-butanone (1.5 mL) and completely frozen by liquid nitrogen. Then CuBr (6.7 mg, 0.047 mmol) was added into the Schlenk tube, and the mixture is degassed using three freeze-pump-thaw-cycles, backfilled with argon and stirred overnight at 50°C. To end the polymerization the tube is cooled and opened, the reaction solution was diluted with THF and filtrated over aluminium oxide to remove all copper species. The reaction solution was concentrated by rotary evaporation and then precipitated in cooled n-hexane to obtain BCP-A (10) after vacuum drying.
The 1 H NMR result of BCP-A is showed in Figure S3.

Synthesis of block copolymer B (BCP-B)
All the reaction schemes for the preparation of block copolymers B (BCP-B) are showed in Figure S4.
After reaction, the mixture was extracted with diethyl ether, then dried over anhydrous magnesium sulfate and removed by rotary evaporation sequentially to obtain the raw product of photo-crosslinker B (8)

Synthesis of the BCP-B (15)
BCP-B was synthesized according to our previous published approach by ATRP [1,2] . were mixed in a Schlenk tube with a stirring bar. The compounds were dissolved in 2-butanone (1.5 mL) and completely frozen by liquid nitrogen. Then CuBr (6.7 mg, 0.047 mmol) was added into the Schlenk tube, and the mixture is degassed using three freeze-pump-thaw-cycles, backfilled with argon and stirred overnight at 50°C. To end the polymerization the tube is cooled and opened, the reaction solution was diluted with THF and filtrated over aluminium oxide to remove all copper species. The reaction solution was concentrated by rotary evaporation and then precipitated in cooled n-hexane to obtain BCP-B (15) after vacuum drying.
Yield: 87 % The 1 H NMR result of BCP-B is showed in Figure S5.

Psome B (AAF-Psome B (HFF B1-2) and (HFF B1-3)) through approaches HFF 2 and 3 and fluorescence study
The fluorescence intensity of AAF-Psome B (HFF B1-2) after purification HFF B1-2 decreases from around 2.8×10 6 (AAF-Psome B (HFF B1)) to around 1. The fluorescence intensity of AAF-Psome B (HFF B1-3) decreases only from around 1.9×10 6 (AAF-Psome B (HFF B1-2)) to around 1.7×10 6 . A low reduction (< 2%) of loading efficiency for avidin-Alexa Flour 488 conjugates (15.5 ± 1.8%) is obtained. Obviously, only a tiny part of avidin-Alexa Flour 488 conjugates is released from Psome B during HFF 3 purification ( Figure   3). We hypothesize that the avidin-Alexa Flour 488 conjugates is mainly released from hydrophobic membrane (location 3) at highly swollen membrane and that after 3-times applied HFF the most avidin-Alexa Flour 488 conjugates is located at the lumen and inner hydrophilic shell of membrane and less of avidin-Alexa Flour 488 conjugates in the hydrophobic membrane ( Figure 3). Figure Captions and Tables   Table S1. The composition, molecular weight and dispersity (Ð) of BCP-A. 1 Determined by 1 H-NMR Spectroscopy, 2 Determined by GPC.   the key factor for the cargo uptake into pH-responsive polymersome. No significant differences between linear and spherical cargo in the uptake can be concluded from recent and previous results. [4] The effect of surface charge is slight, but it is difficult to generalize due to retarding properties of cationic avidin biomacromolecules in protonated polymersome membrane in recent and previous results. [4] Thus, the results of sequential uptake of Biotin-PEG3kDa and Biotin-HRP indicate the uptake in molecular weight-dependent manner, not shape-or surface charge-dependent manner.                        Compared to the Psome B without Avidin, the molar masses are lower and radii are higher due to integration of Avidin. In case of in-situ loaded Avidin-Psome B, a certain free amount is detected in RI signal due Avidin excess ( Figure S25d). Obviously, a certain amount of Avidin biomacromolecules are located on the surface of the membrane. After the purification of Avidin-Psome B by HFF, the free Avidin biomacromolecules in the solution and from the membrane surface are removed ( Figure S25e) and a decrease of the scaling parameter  from 0.41 to 0.35 is observed ( Figure S27a). The conformation changes from irregular, compact shape to more spherical architecture after the removal of Avidin biomacromolecules from the membrane surface. Furthermore, the decrease of density can be explained with the purification step. Figure S28.  parameter (Rg/Rh) vs molar masses of Avidin-Psome B, HFF purified with different Biotin-PEG3kDa loading conditions (pH 5.0…red; pH 6.0…green; pH 7.0…blue) determined by AF4. [2] D. Gräfe, J. Gaitzsch, D. Appelhans, B. Voit, Nanoscale 2014, 6, 10752.