Block Length-Dependent Protein Fouling on Poly(2-oxazoline)-Based Polymersomes: Influence on Macrophage Association and Circulation Behavior

Polymersomes are vesicular structures self-assembled from amphiphilic block copolymers and are considered an alternative to liposomes for applications in drug delivery, immunotherapy, biosensing, and as nanoreactors and artificial organelles. However, the limited availability of systematic stability, protein fouling (protein corona formation), and blood circulation studies hampers their clinical translation. Poly(2-oxazoline)s (POx) are valuable antifouling hydrophilic polymers that can replace the current gold-standard, poly(ethylene glycol) (PEG), yet investigations of POx functionality on nanoparticles are relatively sparse. Herein, a systematic study is reported of the structural, dynamic and antifouling properties of polymersomes made of poly(2-methyl-2-oxazoline)-block-poly(dimethylsiloxane)-block-poly(2-methyl-2-oxazoline) (PMOXA-b-PDMS-b-PMOXA). The study relates in vitro antifouling performance of the polymersomes to atomistic molecular dynamics simulations of polymersome membrane hydration behavior. These observations support the experimentally demonstrated benefit of maximizing the length of PMOXA (degree of polymerization (DP) > 6) while keeping PDMS at a minimal length that still provides sufficient membrane stability (DP > 19). In vitro macrophage association and in vivo blood circulation evaluation of polymersomes in zebrafish embryos corroborate these findings. They further suggest that single copolymer presentation on polymersomes is outperformed by blends of varied copolymer lengths. This study helps to rationalize design rules for stable and low-fouling polymersomes for future medical applications.


Materials and Methods
All the data was plotted using GraphPad Prism 9.0.0.

Polymersome Formation
PMOXA-b-PDMS-b-PMOXA block copolymers from our previous studies [1][2][3] and two commercial block copolymers (Polymer Source, P18140D-MOXZDMSMOXZ (6-65-6), Mw: 500-4800-500 g/mol, Đ = 1.3 and P18140A-MOXZDMSMOXZ (21-65-21), Mw: 1800-4800-1800 g/mol, Đ = 1.35) were used.First, the copolymers were dissolved in ethanol at 20 mg/mL using a bath sonicator.The desired amount was transferred to glass vials, and ethanol evaporated by hand using a nitrogen stream until dryness.The thin film was then hydrated in phosphate buffered saline (PBS, Sigma-Aldrich, D8537) and stirred for at least overnight at RT.The solution was then extruded through 0.1 µm polycarbonate membranes at least 15 times (Avanti, Sigma-Aldrich, 610000).Subsequent purification was performed using PD Minitrap or PD MidiTrap columns (GE Healthcare) equilibrated in PBS.SRB loading was performed by hydration of the thin film with 1 mM SRB in PBS and following the same steps as above but using two columns sequentially to purify away all the free dye.Encapsulation was quantified by measuring fluorescence of the purified samples in a plate reader (SpectraMax M5, Molecular Devices).To label the membrane of polymersomes, 1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindodicarbocyanine Perchlorate (0.5 mol%, DiD, Thermo Fisher Scientific, D307) was added to the copolymer ethanol stocks before forming the films.After running through a PD MidiTrap column equilibrated in PBS, the fluorescence of the polymersomes was measured on a plate reader (SpectraMax M5, Molecular Devices), which also allowed adjusting all the samples to the same amount of fluorescence for cell uptake studies (see below).Samples were sterilized by means of filtration through a 0.22 µm syringe filter (Millipore, SLGV013SL) inside a biosafety cabinet and subsequently stored at 4 °C.
Giant polymersomes at the microscale were formed by the spontaneous hydration method. [4]iefly, a 7 mL glass vial was first plasma treated for 1 min, then a small volume (40 µL) of copolymer stock solution in ethanol at 6 mg/mL was added and dispersed on the vial bottom and the ethanol was allowed to evaporate slowly.Once dried, the film was further dried in a vacuum desiccator for 5 h.Then the film was hydrated with 0.6 mL 0.3 M sucrose and the vial sealed with parafilm and incubated without agitation at 60 °C overnight.The next day the giant polymersomes were pipetted away from the glass vial bottom and used unpurified.

DLS and Zeta Potential Measurements
Measurements (n = 3) were conducted on a Malvern Zetasizer Nano-ZS.70 µL of polymersome solution in PBS was pipetted into single use microcuvettes for DLS.For zeta potential measurements, 950 µL of 0.3 M sucrose was mixed with 50 µL polymersome samples in PBS and transferred to zeta potential cuvettes.

TEM and Cryo-TEM
After plasma cleaning TEM grids (Electron Microscopy Sciences, CF200-Cu, 215-412-8400) for 1 min, 5 µL of polymersome solution in PBS (0.5 mg/mL) was added, incubated for 1 min and blotted away.Sample addition was followed by two ddH2O drop washes before negatively staining the sample with two drops of 2 wt% uranyl acetate in water (0.45 µm filtered), whilst leaving the second drop on the grid for 15 s before blotting away.The overnight dried grids were then imaged on a JEOL 2100F.
For cryo-TEM, a glow-discharged Quantifoil R2/2 grid (400 copper mesh, carbon coated, Quantifoil Micro Tools GmbH, Großlöbichau, Germany) was loaded with 3 μL of polymersome solution (8 mg/mL) in PBS and incubated for 10 s, blotted away for 4 s and plunged into liquid ethane using an automatic plunge freezer FEI Vitrobot (Thermo Fisher Scientific, Waltham, MA, USA) operated at 21 °C and a 100% relative humidity.The samples were then imaged on a JEOL JEM-2100f (JEOL Ltd., Tokyo, Japan) equipped with a TVIPS TemCam-XF416 CMOS camera (Tietz Video and Image Processing Systems GmbH, Gauting, Germany).

Confocal Fluorescence Imaging
First 280 µL of a 1/30 dilution of the FBS-OG488 stock in PBS (finally a 1/900 dilution of FBS) was transferred to each well of an 8-well ibidi plate (80827, ibidi, Germany).Then, 20 µL of the giant polymersome stock in 0.3 M sucrose was added and the plate was incubated static at 37 °C for 4 h.Then, 20 µL of 100 µM Bodipy630 (Thermo Fisher Scientific, D10000, NHS ester deactivated by overnight incubation in PBS) in PBS was added to visualize the membrane before imaging the samples using a commercial LSM 880 (Carl Zeiss, Jena, Germany).Three random spots were chosen per sample for imaging and all the imaging conditions were kept the same for all samples.The Bodipy630 red channel and phase channel images were brightness adjusted in Fiji to optimize visualization of giant location, while the FBS-OG488 channel images were left unmodified.

FCS Measurements
Polymersome samples in PBS (4 mg/mL) were first diluted 1/10 in a 1/20 dilution of the FBS-OG stock in PBS (finally 1/600 FBS dilution) and incubated at 37 °C, 450 RPM using a ThermoMixer.For the clusterin binding experiment, polymersome samples in PBS (4 mg/mL) were mixed 1 to 1 with clusterin-OG stock (see above, 33 µg/mL in PBS) and incubated at 37 °C for 7 h, 450 RPM using a ThermoMixer.Aliquots of 5 µL were taken to conduct FCS measurements on a commercial LSM 880 (Carl Zeiss, Jena, Germany) equilibrated at 37 °C, with data analysis performed with PyCorrfit program 1.1.6. [5]A dilution series of free OG488 in PBS (D = 5.49 x 10 -6 cm 2 /s at 37 °C, D = 4.1 x 10 -6 cm 2 /s at 25 °C) [6] was employed to calibrate and yield the x-y dimension of the confocal volume ( !" # ), which is necessary in order to calculate diffusion coefficients (D) for all the samples and subsequent transformation to hydrodynamic diameters using the Stokes-Einstein equation. .

SPARTA®
SPARTA® has been described elsewhere. [7]We herein used a custom confocal Raman microspectroscope assembled on a Cerna platform (Thorlabs, UK).The components included a spectrograph (HoloSpec-F/1.8-NIR,Andor, UK) and an iDus 416ALDC-DD (Andor, UK) thermoelectrically cooled (−60 °C) backilluminated CCD camera.The 63×/1.0 NA WI objective (W Plan-Aprochromat, Zeiss, Oberkochen, Germany) was directly placed into the polymersome PBS solution and a Raman excitation laser at 785 nm (200 mW, Cheetah, Sacher Laser Technik, Germany) was employed.Many excitation wavelengths can be used for Raman scattering.While shorter wavelength excitation offers increased Raman signal, here we selected a longer wavelength to reduce potential laser burning of the sample, or sample photoluminescence.Each trapped particle was measured for 20 s, before disabling the laser for 1 s to release the trapped polymersome and allow a new polymersome to diffuse into the trap.
Blank PBS measurements were used for background subtraction.Custom MATLAB scripts for cosmic spike removal, spectral response correction (785 nm reference standard, National Institute of Standards and Technology, US), background subtraction, baseline correction, smoothing, and normalization were employed for Raman spectra analysis.PCA analysis was performed using the PLS ToolBox Version 8.8.1 (Eigenvector Research, Inc. WA, USA).

Simulations Details
All-atom classical molecular dynamics (MD) simulations were performed using the graphical processing unit (GPU) accelerated NAMD (version 2.13) software [8] with the CGENFF36 force-field. [9,10]Charges and atom types for the POx ligands were obtained via analogy and additional bond, angle and dihedral parameters for the attachment ligands to the surface and within the POx ligands were generated using the ffTK tool in VMD. [11]All simulations were conducted in the isothermal-isobaric ensemble (NPT) at 310K using a Langevin thermostat with a damping coefficient of 1 ps -1 ; whereas the Nosé-Hoover Langevin piston pressure control (with piston oscillation period of 0.2 ps and piston decay time of 0.1 ps) provided a constant atmospheric pressure environment. [12,13]Long range electrostatic interactions were calculated by the particle-mesh Ewald method with a grid spacing of 1.0 Å.A cut-off radius of 12 Å was used for the short range (i.e.Lennard-Jones) interaction with a switching implemented at 10 Å.
Each system was run for 50 ns of production MD for analyses after 50 ns of equilibration.
Membrane construction: In order to construct membranes from the PMOXA-b-PDMS-b-PMOXA triblock polymers, the chains were aligned parallel in a square formation.We first restrained the x-y motion of the nitrogen atoms within the PMOXA monomers as well as the terminal silicon atoms in the PDMS.We then slowly reduced the z-position of each group, without pressure coupling, in order to compress the membrane to a realistic thickness from the experimental thicknesses taken from Itel et al. [1] Once compressed, we ran NPT equilibration on the system for 50 ns using the same parameters as above.

Cell Interaction Study by Imaging and Flow Cytometry
RAW 264.7 cell line was cultured in DMEM medium (high glucose) containing 10% (v/v) FBS, P/S (1% (v/v), Sigma-Aldrich) and L-glutamine (1% (v/v), Sigma-Aldrich, G7513, now called full medium).Briefly, 1 mL 300 000 RAW 264.7 cells/mL per well were seeded into 24 wellplates (for flow cytometry) or 0.8 mL 300 000 RAW 264.7 cells/mL in 4 well ibidi plates (ibidi, Germany, for imaging).After incubation for 24 h at 37 °C, the spent medium was replaced with fresh full medium and polymersome solution in PBS using PBS/media ratios of 1/4 or 1/10.All The sample droplet was deposited in an ibidi 8-well plate (80827, ibidi, Germany) and excited with a 488 nm Ar + laser focused 200 µm above the glass plate.Fluorescence intensity fluctuations were recorded through a 40x C-Apochromat water immersion objective (NA 1.2) and applying appropriate filter sets.25 intensity traces of 5 s each were recorded and autocorrelated per sample, whilst the shown auto-correlation curves represent the average curves of the whole measurement of 125 s.FBS-OG or clusterin-OG data was first fitted with one component fits  $%&'( () and all the data was subsequently analyzed with two-component fits  #%&'( (), fixing the diffusion time  ) of the first component to the obtained free FBS-OG or clusterin-OG diffusion time and the second component to a diffusion time corresponding to 100 nm diameter nanoparticles ( $ ,  # ).For 3-19-3, the second component ( # ) was not fixed, due to aggregation.A triplet fraction T with corresponding triplet time  *+,( was included and fixed between 1 -10 µs and the structural parameter , which is the ratio of height to width of the confocal volume was set to 5. The fits yielded the corresponding fractions ( $ ,  # ;  = 1 + 2) with F2 *100 being plotted as particle fraction.All the data points (n = 25) for all the three independent experiments (N = 3) are shown in Figure S6 and S7, while the medians for the three independent experiments are summarized in Figure 2c,d. $%&'( ()

Figure S2 .
Figure S2.Polymersome stability.(a,b) DLS peak values (intensity distribution) for various polymersomes incubated at 37 °C in PBS (mean ± s.d. of technical triplicates).(c) DLS measurement (intensity distributions) of assemblies made from various copolymers (mean of technical triplicates) after incubation in PBS or 10 v/v% FBS (F) at 37 °C, after mixing and 22 h later.(d) SPARTA® data (from Figure 1h) showing Raman peak intensity at 1030 and 1490 cm -1 Raman shift for all the traps (n > 200 traps each).All data was normalized to the main PDMS peak (Si-C at 708 cm -1 ) to compare the variations in PMOXA content.

Figure S6 .
Figure S6.FCS analysis of protein fouling on polymersomes.Normalized average autocorrelation curves from FCS measurements with FBS-OG488 mixed with unlabeled polymersomes (two-component fits) and controls of free FBS-OG488 and OG488 dye alone.(b-d) 3 independent experimental repeats of particle fraction for two-component fits of FCS curves (as shown in (a)) revealing extent of protein fouling on polymersome surface (n = 25 technical repeats each).Box-plots: Center line, the median; box limits, upper and lower quartiles; whiskers, minimum and maximum values.Median values from these 3 independent experiments are summarized in Figure 2c.

Figure S7 .
Figure S7.FCS analysis of protein fouling and clusterin binding on polymersomes made from copolymer blends of 6-65-6 and 21-65-21.(a-c) 3 independent experimental repeats of particle fraction for two-component fits of FCS curves (as shown in Figure S6a) revealing extent of protein fouling on polymersome surface (n = 25 technical repeats each).(d) Particle fraction for two-component fits of FCS curves revealing the extent of clusterin-OG488 binding on polymersome surfaces after incubation for 7 h (n = 25 technical repeats each).Box-plots: Center line, the median; box limits, upper and lower quartiles; whiskers, minimum and maximum values.Median values from these 3 independent experiments are summarized in Figure 2d.