Metabolically Active, Fully Hydrolysable Polymersomes

Abstract The synthesis and aqueous self‐assembly of a new class of amphiphilic aliphatic polyesters are presented. These AB block polyesters comprise polycaprolactone (hydrophobe) and an alternating polyester from succinic acid and an ether‐substituted epoxide (hydrophile). They self‐assemble into biodegradable polymersomes capable of entering cells. Their degradation products are bioactive, giving rise to differentiated cellular responses inducing stromal cell proliferation and macrophage apoptosis. Both effects emerge only when the copolymers enter cells as polymersomes and their magnitudes are size dependent.

Characterization: SEC. The polyesters were dissolved in SEC grade THF and filtered through a 0.2 μm syringe filter prior to analysis. SEC, Shimadzu LC-20AD, was used to characterize the molecular weights and dispersities. The instrument was equipped with a refractive index (RI) detector and two Mixed Bed PSS SDV linear S columns. THF was used as the eluent, at a flow rate of 1.0 mL/min at 30 °C. Narrow molar mass polystyrene standards were used to calibrate the instrument.

NMR.
All NMR Spectra were recorded using a Bruker AV 400 MHz spectrometer.

DLS.
Dynamic light scattering (DLS) was used to determine the hydrodynamic diameter (D h ) and polydispersity of the nanostructures formed from PCL-b-PE, in aqueous solution, and was measured using the Zetasizer Nano series instrument (Malvern Instruments Zen1600). The scattering angle was fixed at 173°. Data processing was carried out using cumulant analysis of the experimental correlation function and the Stokes−Einstein equation was used to calculate the hydrodynamic radii. All solutions were analyzed using disposable polystyrene cuvettes.
TEM. TEM imaging was performed using a JEOL JEM-2200FS microscope, equipped with a field emission gun (FEG) at 200 kV, and an in-column energy Omega filter. The microscope was used in energy-filtered transmission electron microscopy (EFTEM) mode in order to increase image contrast by collecting only elastically scattered electrons. The software used for image acquisition and processing was the Digital Micrograph™ software (version 3.20). Images were recorded using a direct detection camera K2 IS from Gatan working in linear mode. This camera allowed for beam sensitive materials to be imaged at low electron doses. 400 mesh copper grids were glow-discharged for 40 s to render them hydrophilic. Then, 5 µL of polymersome dispersions (concentration ~0.5 mg/mL) were deposited onto the grid for 1 min. The grid was blotted with filter paper and immersed in Uranyless (Delta Microscopies, France) staining solution for 40 s for negative staining. The grid was blotted again and dried under vacuum for 1 min.

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Zeta Potential. Zeta potential studies were conducted from 20 °C to 85 °C using a ZETASIZER Nano series instrument (Malvern Instruments).

Fluorescence Spectroscopy.
Fluorescence experiments were carried out to evaluate the drug-loading efficiency via a Cary eclipse Fluorescence Spectrometer (VARIAN). The drug release profiles were also monitored using fluorescence spectroscopy.

Synthesis of PCL-OH Macroinitiator
The procedure used for the two macro-initiators is illustrated for the synthesis of PCL 38 -OH. The

Procedure for calibrating the quantity of DOX·HCl in PBS solution
The procedure was previously reported using tris buffer. [1] In this work, a stock PBS solution (10.0 mM, pH 7.4) of DOX·HCl (500 µg·mL -1 ) was carefully diluted to a range of concentrations (0.5, 1.0, 2.0, 3.0 and 4.0 µg·mL -1 ) using PBS solution (10.0 mM, pH 7.4). The fluorescence intensities of these samples were measured using the Fluorescence spectrometer. The PMT (photomultiplier tube) voltage was set to be 570 V and the excitation and emission slits were both 5 nm. Emission scans from 500~800 nm were collected, with λ ex fixed at 461 nm. The peak intensities at 591 nm were used to prepare the calibration. Calibration data was linearly fit in Origin 8.1 and is reported in Figure S14B.
The same instrument settings were used for the investigation of drug release profiles.

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Colloidal self-assembly, drug-loading and in vitro release procedures.  The calibration for DOX and RhB emission ( Figure S13a and S14a) was pre-determined and was applied to determine the cumulative release profiles of either DOX (Fig. S13c) or RhB (Fig. S14c) overtime. The burst release was determined by fitting the cumulative release profile for the drugloaded polymersomes against the free drug release across the dialysis membrane.

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All preparations were performed in sterile conditions and in biological safety cabinets (KS15-Thermo Fisher Scientific). The polymer stock solution (2.00 mL, 1.0 mg mL -1 in THF) was exposed to three hours. [2] We chose this PMA concentration as it has been found to not undesirable regulate genes expression. [3] For cell viability, the Thiazolyl Blue Tetrazolium Blue (MTT, Sigma) method was used.
Briefly, cells were seeded at a concentration of 5 x 10 3 cells per well in a 96 well plate overnight (O.N.). Increasing concentrations of polymersomes (398 nm) were then added in the growth media, namely 5.6, 14 and 28 μg mL -1 , for 24 and 48 hours. The medium growth was then removed and an acidified solution of isopropanol was added to dissolve the water-insoluble MTT formazan. The solubilized blue crystals were measured calorimetrically at 570 nm (plate reader ELx800, BioTek).
The cytotoxicity profiles of both polymersomes (398 nm) and degradation products were determined using the same method. The degradation products were obtained by: first, enzymatic hydrolysis of polymersome solutions, then removal of Pseudomonas cepacia lipase using centrifuge filtration (MWCO = 14 kg·mol -1 , which is much lower than the molar mass of Pseudomonas cepacia lipase (~ 34 kg·mol -1 ) to ensure complete removal [4] ). Assuming all mass was recovered from centrifuge BioTek) by reading at 570 nm.

Reverse transcription polymerase chain reaction (RT-PCR), and PCR assays
First, the polymersomes (53 nm), polymersomes (398 nm) and degradation products solutions were diluted to 28 μg mL -1 which is the maximum concentration used in the MTT investigation. Cultured cells, incubated for 24 h with polymersomes (28 μg mL -1 ), were lysed and total RNA was collected by GAPDH and ACTB were used as reference genes.
Quantitative analysis was assessed with QuantiTect SYBR Green RT-qPCR Kit (Qiagen). The amplification process was done in 20 µL/tube, using the following steps: 95 °C for 5 min to make active the DNA polymerase, followed by 40 cycles of 95 °C (10 s) for denaturation, and 60 °C (30 s)

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for combined annealing and extension for all primers. Melting curve was also acquired, to analyze the sample quality, from 55 °C to 99 °C, by increasing of 1 °C min -1 . Data were analysed via ΔΔCt value.

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End group determination to confirm block structure 31 P{ 1 H} NMR was used to monitor the polymer end groups for both PCL-OH and PCL-b-PE. The polymers were reacted excess 2-chloro-4,4,5,5-tetramethyl dioxaphospholane and bisphenol A was used as an internal reference.
The signals at 147.85 ppm and 147.05 ppm are assigned to the primary -OH end groups of the PCL-OH macroinitiator and the -OH end group of PCL-b-PE block copolyester, respectively.
A B S15

Zeta Potential Determination
The block polymer oligo(ethylene glycol) side chains are known to undergo a hydrophilic to hydrophobic transition when the polymer is heated above a particular temperature. [6] This loss of the hydrophilic corona is expected to significantly decrease the colloidal stability of the vesicles. Since the absolute value of the zeta potential is a measure of electrostatic repulsion between neighbouring colloidal nanoparticles and indicates colloidal stability, 22,27,34 the zeta potential of self-assembled nanostructures was monitored from 25 to 90 °C. Usually, colloidal systems having zeta potentials ranging from ±40 mV to ±60 mV are considered as showing 'good stability'. [7] Figure S8   determined by SEC in THF at 30 °C, calibrated using narrow molar mass polystyrene standards. S18 Figure S11. 1 Figure 3B. The cell nuclei were stained with DAPI in blue and far-red CellMask TM was used for cell membrane staining. Figure S16. Viability (MTT) assay of cells incubated at increasing concentrations of degradation products (red), polymersomes (53 nm) (blue), and polymersomes (398 nm) (black) for 24 and 48 hours. All experiments were carried out as three independent replicates, followed by t-test statistical analyses. *p<0.05 and **p<0.01.