Photo‐Cross‐Linked Dual‐Responsive Hollow Capsules Mimicking Cell Membrane for Controllable Cargo Post‐Encapsulation and Release

Multifunctional and responsive hollow capsules are ideal candidates to establish highly sophisticated compartments mimicking cell membranes for controllable bio‐inspired functions. For this purpose pH and temperature dual‐responsive and photo‐cross‐linked hollow capsules, based on silica‐templated layer‐by‐layer approach by using poly(N‐isopropyl acrylamide)‐block‐polymethacrylate) and polyallylamine, have been prepared to use them for the subsequent and easily available post‐encapsulation process of protein‐like macromolecules at room temperature and pH 7.4 and their controllable release triggered by stimuli. The uptake and release properties of the hollow capsules for cargos are highly affected by changes in the external stimuli temperature (25, 37, or 45 °C) and internal stimuli pH of the phosphate‐containing buffer solution (5.5 or 7.4), by the degree of photo‐cross‐linking, and the size of cargo. The photo‐cross‐linked and dual stimuli‐responsive hollow capsules with different membrane permeability can be considered as attractive material for mimicking cell functions triggered by controllable uptake and release of different up to 11 nm sized biomolecules.

. 1 H-NMR spectrum (DMSO-d6) of PNMB11 with all peaks labeled to the corresponding groups in the molecule. 1 H NMR signals of NIPAM at 3.84 ppm, BMA at 6.3-7.7 ppm, and MA at 12-12.75 ppm, respectively. 14 Figure S2. A) Transmittance curve upon heating for LCST measurements of block copolymer PNMB21, PNMB11, PNMB12, and PNMB14 at pH 7.5 (UV-vis spectroscopy). B) Reversible volume phase transition of PNMB11 upon switching between 25 ℃ and 45 ℃ at pH 7. (open symbol=25 ℃; solid symbol=45 ℃). 15 Figure S3. Heating curves of the LCST measurements of random polymer PRMNB, block copolymer PNMB11 and homopolymer PNIPAM at pH 7.5, followed by transmittance (UV spectroscopy). 16 Figure S4. Heating curves of the LCST measurements of block copolymer PNMB11 at pH 5.5, pH 6.5, pH 7.5, pH 8.5 and pH 9.5, respectively, followed by transmittance (UV-vis spectroscopy). 17 Figure S6. Reversible swelling-shrinking of multilayer films [PAH/PNMB11] 3 /PAH/PSS on planar substrate after photo-crosslinking for 10 min (A) and 20 min (B) upon switching between temperature 25℃ and 45℃ at pH 5.5, pH 7.4 and pH 8.5 buffer, respectively (open symbol=25 ℃; solid symbol=45 ℃) (n=3). 18 Table S1. Swelling ratio of multilayer films [PAH/PNMB11] 3    BMA, can be found in the polymer chain, indicating copolymerization took place. Furthermore, the ratio of MA and NIPAM in the copolymer chain was calculated by the integration of the ratio between the specific signals of MA at 12-12.75 ppm and NIPAM at 3.84 ppm, respectively, and the content of PBMA was calculated to be about 5% by the integration of the ratio between the specific signals of BMA (6.3-7.7 ppm) and PNIPAM (3.84 ppm), respectively.
UV/vis spectroscopy. The lower critical solution temperature (LCST) of the polymers was measured on a Tepper TP1 photometer (Mainz, Germany). Transmittance of the polymer in MilliQ water at 670 nm was monitored as a function of temperature (cell path length: 12 mm; one heating/cooling cycle at a rate of 1 °C min -1 ) and the critical temperature was determined at 50% of relative transmittance.
Zeta potential. Zeta potential of the polyelectrolytes PAH and PNMB11 was determined using Zetasizer Nano-series instrument (Malvern Instruments, UK) equipped with a multi-purpose autotitrator. PAH (1 mg mL -1 ) without salt (pH adjusted with HCl to pH 4) was titrated with NaOH solution and PNMB (1 mg mL -1 ) without salt (pH adjusted with NaOH to pH 9.5) was titrated with HCl solution.
Zeta potential values of the bare and multilayered silica particles were measured on a

ZETASIZER Nano series instrument (Malvern Instruments, UK) with a Dispersion Technology
Software (version 7.10). The data was collected by three repeated experiments at 25 ℃ with a voltage of 150V.
Photo-crosslinking of multilayers. UV irradiation of polymer (multi)layers was performed using UVACUBE100 (honle UV Technologies, Germany) equipped with a low intensity (0.1 Wcm -2 ) iron lamp as UV source.
Ellipsometry. The film thicknesses measurements were performed on a UVISEL spectroscopic ellipsometer (HORIBA Jobin Yvon S.A.S, Chilly Mazarin, France). Spectroscopic data was acquired between 400 and 800 nm with a 2 nm increment, and unless otherwise stated, thicknesses were extracted with the integrated software by fitting with a classical wavelength dispersion model. No less than three measurements were taken on each sample.
Dynamic light scattering. The hydrodynamic size of capsules were determined by dynamic light scattering (DLS) using Zetasizer Nano-series instrument (Malvern Instruments, UK) equipped with a 633 nm He-Ne laser at fixed scattering angle of 173°. The data were analyzed using software version 6.12.
Transmission electron microscopy. The diameters and morphologies of the capsules were observed using a transmission electron microscopy (TEM) Libra 120 equipped with a charge coupled device (CCD) camera at an accelerating voltage of 120 kV. 5 µ L of the capsules or particles were dispersed in water with the concentration of 1 mg mL -1 and allowed to adsorb for 5 min. onto a 300 mesh, carbon film coated copper grid, and the specimen was dried at room temperature or the grid was blotted dry using filter paper.
SEM samples were prepared from 1 µL of a concentrated capsule solution on Si wafers which were sputter-coated with gold and were analyzed using a Zeiss Ultra 55 Gemini scanning electron microscope.

Synthesis of PRMNB random copolymer
PRMNB random copolymer was synthesized by RAFT polymerization, as follows: RAFT liquid nitrogen was carried out. The 1,4-dioxane-containing monomer/polymer mixture was twice poured in diethylether to precipitate the desired PRMNB random copolymer, followed by the removal of the random copolymer by filtration and dried under suction. Yield: 75 %.

Preparation of Multilayers Assembly on Planar Substrates
Multilayers were deposited on Si wafer substrate by using LbL technique. The polyelectrolytes PAH and PNMB11 were prepared at a concentration of 1 mg mL -1 in 0.4 M NaCl and the pH of the solutions was adjusted to 6.0, 6.5, 7.0, 7

Reversible swelling-shrinking behavior of the crosslinked multilayer assembly films on planar substrates
The polymer-coated Si wafers with different crosslinking time were incubated with phosphate buffer with different pH values 5.5, 7.4, and 8.5, respectively. At each pH state, polymer-coated Si wafers were allowed to stand for 30 min. Then the environmental temperature for the Si wafers was switched between 25 ℃ and 45 ℃ and the film thickness was determined by ellipsometric measurements. This process was repeated for several cycles as shown in the main text. No less than 20 measurements were taken on each temperature.

Reversible swelling-shrinking behavior of the crosslinked hollow capsules
The photo-crosslinked hollow capsules prepared by different crosslinking period were incubated with phosphate buffer possessing pH 5.5 and 7.4, respectively. At each pH state, hollow capsules were allowed to stand for 20 min. Then the temperature of the capsules was switched between 25 ℃ and 45 ℃ and the particles diameters were determined by DLS. This process was repeated for several cycles as shown in the main text. No less than 20 measurements were taken on each temperature. nm) were synthesized and characterized as previously described in reference. [1] To detect PEI-Mal nanoparticles in the following experiments, they were labeled with a UV-vis active dye rhodamine B. We labeled these molecules as follows: rhodamine B (1.0 mg) was dissolved in DMSO (0.2 ml). Then, PEI-Mal 5 or PEI-Mal 25 (50 mg), respectively, was dissolved in deionized water (1 mL). Both solutions were mixed and stirred overnight. Non-bound rhodamine B was removed by dialysis method. Powders of the materials were obtained by freeze drying process.

Encapsulation of PEI-Mal 5 and PEI-Mal 25
A concentrated solution containing the capsules with 5 mol-% crosslinker and photo-

Turbidity study on block and random copolymers
The 1:1 block copolymer PNMB11 possessed a good thermo-sensitivity as found for PNIPAM homopolymers over a broad pH range up to pH 8.5 (above pH 9 the aggregated block copolymer was highly swollen owing to ionization of carboxyl groups ( Figure S4)). However, the cloud point of the respective 1:1 random copolymer PRMNB disappeared already at pH 7.5 ( Figure S3 in the Supporting Information), because the MA components (which are ionized at pH 7.5) convey sufficient solubility to offset the aggregation of hydrophobic temperature-sensitive components. To investigate the effect of MA content on the LCST of the block copolymers, turbidity measurements were performed ( Figure S2A). It was observed that the LCST values of block copolymer rapidly rose with the molar fraction of MA in the block copolymer, with the cloud point increasing from 39.9 ℃ for the 2: 1 ratio of NIPAM and MA in PNMB21 to 49.0 ℃ for the 1: 4 ratio of NIPAM and MA in PNMB14 due to the hydrophilic character of the MA monomers in the polymeric block of MA and BMA. Increasing the MA content in PNMB ( Figure   S2A), the degree of hydrogen-bond-driven interactions in PNMB solution is higher than in pure PNIPAM homopolymer solution. Thus, there is a need for more energy to destroy the hydrogenbond interactions which causes an increase in their LCST behavior. [2,3]     pH 5.5 pH 6.5 pH 7.5 pH 8.5 pH 9.5 Figure S4. Heating curves of the LCST measurements of block copolymer PNMB11 at pH 5.5, pH 6.5, pH 7.5, pH 8.5 and pH 9.5, respectively, followed by transmittance (UV-vis spectroscopy).   Swelling ratio between below (25 ℃) and above LCST (45 ℃) a) Swelling ratio between pH 8.5 and pH 5.