Biomimetic Hybrid Nanocontainers with Selective Permeability

Abstract Chemistry plays a crucial role in creating synthetic analogues of biomacromolecular structures. Of particular scientific and technological interest are biomimetic vesicles that are inspired by natural membrane compartments and organelles but avoid their drawbacks, such as membrane instability and limited control over cargo transport across the boundaries. In this study, completely synthetic vesicles were developed from stable polymeric walls and easy‐to‐engineer membrane DNA nanopores. The hybrid nanocontainers feature selective permeability and permit the transport of organic molecules of 1.5 nm size. Larger enzymes (ca. 5 nm) can be encapsulated and retained within the vesicles yet remain catalytically active. The hybrid structures constitute a new type of enzymatic nanoreactor. The high tunability of the polymeric vesicles and DNA pores will be key in tailoring the nanocontainers for applications in drug delivery, bioimaging, biocatalysis, and cell mimicry.


Synthesis of PMPC 25 -PDPA 72 by ATRP
Block copolymer PMPC-PDPA was synthesized by atom-transfer radical-polymerization (ATRP) following a published protocol. [1] Briefly, a solution in morpholinoethylbromoisobutyric acid ester (ME-Br, synthesis described previously) [2] (0.190 g, 0.68 mmol, 1 eq.) in EtOH (5 mL) was placed in a round-bottom flask before addition of MPC (5.000 g, 1.70 mmol, 25 eq.). The mixture was stirred and further purged with nitrogen for 30 min and heated to 30 °C. Then, a mixture of bpy (0.223 g, 1.42 mmol, 2 eq.) and Cu(I)Br (0.097 g, 0.68 mmol, 1 eq.) was added under a constant nitrogen flow. The mixture was stirred for 60 min to yield a highly viscous brown substance and sampled with NMR to estimate the extent of conversion. Meanwhile, a solution of DPA (12.27 g, 57.6 mmol, 85 eq.) in EtOH (13 mL) was prepared and purged with nitrogen for 60 min in a separate flask. DPA solution was added to the polymerization mixture, and the reaction solution was purged for another 10 min

Synthesis of PDPA 70 -PMPC 25 -S-S-PMPC 25 -PDPA 70 by ATRP
A similar synthetic procedure to PMPC 25 -PDPA 72 was used, but with BiBOE 2 S 2 as initiator. [3] Briefly, a solution of BiBOE 2 S 2 (0.1850 g, 0.40 mmol, 1 eq.) in anhydrous EtOH (4 mL with an autosampler, a pressure pump, an FLD-fluorescence emission detector, and a Jupiter 5 µm C18 column from Phenomenex. The mobile phase was composed of (A) water (0.05% TFA) and (B) MeOH (0.05% TFA) using a gradient of 5 -80% B over 120 min. The flow rate was 1 mL min -1 , the detection was monitored at an absorption wavelength of 568 nm, and the injection volume was 20 µL. The reaction achieved full conversion to Cy3-PMPC 25 -PDPA 70 and no free dye was detected.

Preparation of Polymersomes
All polymersome dispersions were prepared by thin film hydration. [4] Typically, block N agg is defined in equation (2) as the ratio between the volume of the polymersome hydrophobic membrane, V p , divided by the product of molecular volume of the PDPA block of the polymer, V PDPA , assuming a vesicle packing parameter p = 1.
The molecular volume , V PDPA , is defined as: where M PDPA is the molar mass of the PDPA block ρ PDPA the bulk density of the hydrophobic chain and is 1.02 g/ml and, N A , is the Avogadro number.

Assembly and Gel Characterization of DNA Nanopores
DNA nanopores with cholesterol tags (NP-3C) and without cholesterol tags (NP-0C) (2D map in Figure S1) were assembled from an equimolar mixture of six DNA strands with sequences listed in Table S1. For NP-0C, oligonucleotides 1-6 were used, and for NP-3C the

AFM Analysis of DNA Nanopores
DNA nanopores NP-0C were analyzed by AFM following a published approach that involves adsorption using divalent cations (NiCl 2 ) [5][6]  Imaging was performed at a setpoint of 0.014 Volts (approximately 60 pN) and at a PeakForce frequency of 2 kHz with a 5 nm PeakForce amplitude. Images were processed using Gwyddion [7] for line-by-line flattening and removal of tilt using a first order polynomial, and the color scale was set to 1.8 nm to show individual nanopores against the background ( Figure 2B, Figure S6-A). Line profiles were taken in Gwyddion along the long (length) and short (width) axis of NP-0C as shown by the grey and green lines in Figure S6-A, and plotted in Origin (OriginLab) ( Figure S6-B). The dimensions of the nanopore were determined by this method as the full-width-at-half-maximum (FWHM) of a Gaussian peak fitted to the profiles.
Singular pores were selected for statistical analysis by elevation using Gwyddion ( Figure S6-C). The grain distribution function was used to determine the width (minimum bounding size) and height (maximum bounding size) of the selected nanopores and plotted in Origin ( Figure   2B, Figure S6-D).

Dynamic Light Scattering Analysis of Polymersomes
The Correlogram analysis was performed with software from Malvern. The particle size distribution was calculated by the cumulant analysis method.

Transmission Electron Microscopy of DNA Nanopores and Polymersomes
The (STEM) imaging, [1] using the JEOL 2100 microscope. Polymersomes stained with PTA were first imaged by conventional TEM, followed by analysis in STEM using the dark-field mode to map the distribution of tungsten within the vesicle. Plot profiles were then taken with Image J across the membrane and the FWHM was taken.

Fluorescence Measurements of DNA Nanopores and Polymersomes
Fluorescent measurements were carried out on a Varian Carry Eclipse spectrophotometer.

Chromatography and Characterization by UV-vis Spectroscopy
Porcine trypsin (1000-2000 U/mg) was encapsulated into polymersomes by electroporation following a published procedure. [8] Briefly Polymersomes with encapsulated trypsin were purified from excess free trypsin by SEC using Sepharose 4B. The gel filtration medium (20% slurry in EtOH) was first washed multiple times with PBS and centrifuged (5000 RCF, 5 min). Sepharose was then packed into the chromatographic column and washed 5 times with PBS. The sample (400 µL) was applied to the column, and purified material was eluted by collecting 500 µL fractions.
Isolated trypsin and polymersomes that was not exposed to trypsin were also purified to obtain the corresponding reference elution volumes (10 -15 mL and 5 -6 mL, respectively).
The protein content in the SEC-purified polymersome fraction containing encapsulated trypsin was analyzed by UV-vis spectroscopy to obtain absorbance readings at 280 nm using a Carry Eclipse Varian spectrophotometer. To determine the PMPC 25 -PDPA 72 polymer content, protein-free polymersomes that had been subjected to the same purification procedure as polymersomes with encapsulated trypsin were analyzed by UV-spectroscopy at 220 nm. The polymersome solution (20 µL) was diluted 10-fold in PBS at pH 2.0. The concentration of trypsin was PMPC 25 -PDPA 72 was calculated from absorbance readings and calibration curves for PMPC 25 -PDPA 72 polymer and trypsin in PBS at pH 2.0. The polymer concentration was subsequently used to calculate the number of polymersomes as described in section 1.5.
Absorbance was recorded at 220 nm.
[Poly] (mg.mL -1 ) 1 All values correspond to the concentration in nanoreactor assay mixture, as described in section 1.12. 2 The polymer concentration in the polymersome sample that was not subjected to electroporation was 0.16 mg mL -1 . The concentration was obtained from the absorbance reading of 0.06 at 220 nm ( Figure S16) and a calibration curve ( Figure S18). 3 The number of polymersomes was calculated from the polymersome size (DLS) and the polymer concentration, as described in section 1.5. 4 The number of DNA nanopores per vesicles only refers to ratio in the incubation mixture but not to the value of membrane-inserted pores. 5 The concentration of trypsin encapsulated within polymersome was calculated from the absorbance reading at 280 nm minus the baseline intensity calculated at 300 nm ( Figure   S16; calibration curve, Figure S17) obtained from the difference between the samples that were and were not electroporated ( Figure S17).