Photo-cross-linked and pH-Switchable Soft Polymer Nanocapsules from Polyglycidyl Ethers

Soft polymer nanocapsules and microgels, which can adapt their shape and, at the same time, sequester and release molecular payloads in response to an external trigger, are a challenging complement to vesicular structures like polymersomes. In this work, we report the synthesis of such capsules by photo-cross-linking of coumarin-substituted polyglycidyl ethers, which we prepared by Williamson etherification of epichlorohydrin (ECH) repeating units with 7-hydroxycoumarin in copolymers with tert-butyl glycidyl ether (tBGE). To control capsule size, we employed the prepolymers in an o/w miniemulsion, where they formed a gel layer at the interface upon irradiation at 365 nm by [2π + 2π] photodimerization of the coumarin groups. Upon irradiation at 254 nm, the reaction could be reversed and the gel wall could be repeatedly disintegrated and rebuilt. We further demonstrated (i) reversible hydrophilization of the gels by hydrolysis of the lactone rings in coumarin dimers as a mechanism to manipulate the permeability of the capsules and (ii) binding functional molecules as amides. Thus, the presented nanogels are remarkably versatile and can be further used as a carrier system.

All reaction mixtures used in irradiation experiments were stirred using a magnetic stirring bar.
UV-Vis spectroscopy.UV-Vis spectra were recorded on a JASCO V-630 spectrophotometer connected to a Huber Pilote-one 'petite fleur' thermostate using silicone oil (temperature range -40 °C to 110 °C) as thermofluid.Absorbance (Abs. in arb.units) was measured in quartz glass cuvettes (d = 10 mm) with a scan speed of 1000 nm•min -1 at fast response (data pitch = 1 nm) at T = 25 °C.Spectra analyses were evaluated using the JASCO spectra manager software (Version 2).Samples of prepolymers were analyzed in MeCN and microgel samples dispersed in H2O or in MeCN.
Infrared spectroscopy.IR spectra were carried out on a ThermoNicolet FT-IR Nexus spectrometer and are recorded on a silicon crystal (ThermoNicolet, Smart SplitPEA).
Transmission maxima are reported in wavenumbers (cm -1 ), only selected intensities are reported.

Differential scanning calorimetry.
The glass transition temperature Tg of prepolymers 1-8 and dried microgels I-IV were analyzed by differential scanning calorimetry (DSC).Samples of 5 -10 mg (weighed on a Sartorius Balance CP2P; accuracy ±1 µg) in aluminum crucibles with lid were measured on a Perkin Elmer DSC 8500 equipped with a controlled liquid nitrogen cooling accessory (CLN2, setpoint: -80 °C / -110 °C).The measurements were performed under nitrogen between -80 °C and 60 °C for 1-3 and between -50 °C and 150 °C for 4-8 and I-IV with a heating rate of 10 K•min -1 for the first and 20 K•min -1 for the second heating cycle.Atomic force microscopy.Samples for AFM measurements were prepared on a silicon wafer.First, the wafer was hydrophilized in a PVA Tepla Plasma System 100 (O2, 5 min, 200 W).Microgel with a concentration of 1 mg/mL was spin coated on the wafer using a Laurell WS-650SZ-6NPP/LITE (1200 rpm, 1 min).AFM images were measured using tapping mode (Agilent 5500 SPM/AFM, Agilent Technologies) under ambient conditions.
The standard silicon cantilevers used had a spring constant of 5 -37 N/m and an oscillation frequency of 96 -175 kHz.Data were processed using Gwyddion software, version 2.44.Nile red staining.For confocal microscopy MG II was stained with Nile red.Therefore, 0.5 mL of a 1 mg•mL -1 aqueous dispersion of MG II was stirred with 5 µL of a 0.3 M solution of Nile red in acetone for 30 minutes at room temperature.To remove excessive Nile red, the stained microgel was dialyzed against water (MWCO: 500 -1000 Da).
Confocal and super-resolution microscopy imaging.Confocal and stimulated emission depletion (STED) microscopies were performed with a Leica SP8 Tandem system, equipped with a 93x glycerin objective.For confocal imaging of microgels, samples were mounted onto cover slips (#1.5, Paul Marienfeld GmbH & Co. KG) and covered with a UV-transmissible 1 mm thick quartz microscope slide (Electron Microscopy Sciences) sealed with oil to prevent evaporation.Afterward, samples were excited with a photodiode 405 (λ = 405 nm, unlabeled) or white light laser (λ = 560 nm, Nile red) and signals were detected with a pinhole of 1 a.u. and a gating of 0.8 -6 ns via a HyD detector (λ = 630 -750 nm, unlabeled; λ = 570 -700 nm, Nile red).For STED microscopy the zoom and resolution were adjusted to acquire a pixel size of approximately 20 nm and the signal was depleted by a pulsed laser with a wavelength of 775 nm.Additional z-resolution was gained by reducing the pinhole to 0.6 a.u. and a phase mask of approximately 30%.Images were subsequently deconvolved with the Huygens Professional software (Scientific Volume Imaging B.V.).

Dynamic light scattering. Size distributions of MG I at different pH were measured by DLS
on a Zetasizer NanoZS (Malvern, UK) at a fixed angle of 173°.Very diluted samples were prepared with HCl and NaOH to adjust the respective pH 5, 7, 9 and 11.Samples were filtered using a 1.2 µm PET filter before measurement.The measurements were performed at 20 °C.
X-ray photoelectron spectroscopy.XPS measurements were performed using a K-Alpha + XPS spectrometer (Thermo Fisher Scientific, East Grinstead, UK).Data acquisition and processing using the Thermo Avantage software is described elsewhere. 1All samples were analyzed using a microfocused, monochromated Al Kα X-ray source (30 -400 µm spot size).
The spectra were fitted with one or more Voigt profiles.For intense peaks and/or peaks clearly evidenced by the peak shape, the binding energy uncertainty was around ±0.1 eV.In case of weak peaks and no direct justification by the peak shape, the uncertainty was set to ±0.2 eV.The analyzer transmission function, Scofield sensitivity factors, and effective attenuation lengths (EALs) for photoelectrons were applied for quantification. 2EALs were calculated using the standard TPP-2M formalism. 3All spectra were referenced to the C 1s peak of hydrocarbon at 285.0 eV binding energy controlled by means of the well-known photoelectron peaks of metallic Cu, Ag, and Au.
The fraction of a monomer x (rx) can be obtained from the integral of a peak ∫H # unique to the monomer divided by the number of contributing nuclei N: The calculation is demonstrated using the 1 H-NMR spectrum of polymer 2 (Figure S2).The unique peak of H 5 of tBGE at δ = 1.16 ppm is used to determine rtBGE: No distinct unique peak of ECH is available, as the RCH2Cl signal H 3 overlaps with the signals of the polymer backbone.Thus, the integral of H [1][2][3][4] has to be corrected by 5 protons, corresponding to the tBGE repeating unit.rECH is calculated using: The ratio of ECH and tBGE in polymer 2 is 1.5 : 1 (60 mol% ECH, 40 mol% tBGE).
The same procedure is used to obtain the molar ratios of CumGE, ECH and tBGE in polymers 4-8 and will be shown at the example of polymer 5 ( 1 H-NMR spectrum: Figure S4).
Again, because the spectrum was normalized to the 9 protons of tBGE's tert-butyl group H 5 , rtBGE = 1.Peak H 11 , which is unique to a single proton of a coumarin-functionalized repeating unit, is used to calculate rCumGE: Again, no distinct ECH peak can be used for direct comparison of the monomers.Due to the additional presence of CumGE repeating units, polymer backbone signals H [1][2][3][4]6 does not only need to be corrected by the 5 protons of one tBGE but also by 5 protons of 0.65 CumGE repeating units. rEH is obtained by: Thus, the ratio of CumGE, ECH and tBGE in polymer 5 is 0.65 : 0.94 : 1 (25 mol% CumGE, 36 mol% ECH, 39 mol% tBGE).

Calculation of the Molecular Weight of p(CumGE-stat-ECH-stat-tBGE) 4 by NMR
The number-average molecular weight Mn,NMR of polymer 4 can be calculated, because protons H 8 of side group (SG) and end group (EG) coumarin moieties give distinct signals at d = 7.48 and 7.42 ppm, respectively, in the1 H-NMR spectrum (Figure S5).With the following equations, the degree of polymerization DPx of each constituent monomer x can be calculated using the same unique 1 H-NMR signals previously used for the calculation of monomer ratios: The combined degree of polymerization for all monomers is 30, while a chain length of 25 was targeted in the initial polymerization.This result is within the general margin of error for Synthesis of MG II-pz.Experimental data can be found in the main text.

Optimization of Microgel Synthesis
Microgel synthesis conditions were optimized by comparing the crosslinking reaction of p(CumGE-stat-ECH-stat-tBGE) 4 in abscence or presence of two different photosensitizers (benzophenone or 2,2-dimethoxy-2-phenylacetophenone -DMPA) in the crosslinking procedure.The crosslinking reaction was performed in a miniemulsion, containing 4 (0.25 g), hexadecane (0.02 g) and photosensitizer (0.05 mmol) in toluene (0.88 g) as organic phase and SDS (0.002 g) dissolved in water (4 g) as aqueous phase.After combining both phases, the miniemulsion was prepared using a Branson Ultrasonifier 450 for 15 min (output control 3, duty cycle 50%).The mixture was irradiated for 3 h at λ = 365 nm under vigorous mechanical stirring.After the reaction, a small sample of the reaction mixture was dried, redissolved in MeCN and investigated using UV-Vis spectrometry (Figure S8).UV-Vis absorption spectra of 7-methoxycoumarin in acetonitrile, serving as a small molecule model compound for polymerized coumaryl glycidyl ether repeating units, were recorded to determine the attenuation coefficients at λ = 295, 318 and 350 nm (Figure S12).To estimate the attenuation of light during photocrosslinking of 5, the concentration of coumarin moieties in the toluene phase of the reaction emulsion must be calculated: The largest nanocapsule of II measured via AFM had a diameter of 400 nm (Figure 8e).