N n n n tetramethylenediamine

Amoxicillin trihydrate (AT), N, N methylene-bis-acrylamide (BIS) (Aladdin industrial corporation), pectin (Sigma-Aldrich Co., Ltd., Burlington, VT, USA), 2-hydroxyethylmethacrylate (2HEMA), pluronic F27/poloxamer 407, ammonium persulfate (APS), N, N, N, N-tetramethylene diamine (TEMED), glacial acetic acid, sodium acetate, and sodium bicarbonate were purchased from commercial sources (China). Acetone and ethanol (AR anhydrous) were acquired from Shanghai Titan Scientific Co., Ltd. (Shanghai, China). Deionized water was available as and when required and utilized throughout.

The soft and stiff polyacrylamide gels were prepared to mimic the cellular environment with different mechanical properties. To produce the desired gels, 40% polyacrylamide (final concentration 8%) (BioRad) and 10 mmol/L HEPES pH 8.5 (Sigma‐Aldrich); the stiff gels were formed using 0.1% N,N'‐methylene‐bis‐acrylamide, while the soft gels used 0.06% N,N'‐methylene‐bis‐acrylamide (BioRad). The solutions were polymerized with 10% ammonium persulfate (Sigma‐Aldrich) and N,N,N',N'‐tetramethylenediamine (Sigma‐Aldrich) for about 30 minutes between sterile glass plates. After polymerization, the plates were separated, and 2.5‐cm‐diameter gel discs were cut and placed into 6‐well culture plates for further experiments.
The gels were activated by the addition of 0.5 mmol/L sulfo‐SANPAH (Sigma‐Aldrich) in solution containing 50 mmol/L HEPES pH 8.5 and 0.5% DMSO on the surface of each gel disc and then exposed to UV light for 30 minutes. The gels were washed three times with 50 mmol/L HEPES pH 8.5 (Sigma‐Aldrich). The gels were then coated with 100µg/ml solution of type I collagen (10 µg/cm²), (Sigma‐Aldrich) in 0.1 M acetic acid and incubated overnight at 4°C. Excess acetic acid was removed before use by washing with HBSS (Thermo Fisher Scientific).

Lipids used in this work are Egg PC l-α-phosphatidylcholine (T_t ≤ 0 °C, EPC), Hydro Soy PC l-α-phosphatidylcholine (T_t = 52 °C, HSPC), cholesterol (Chol), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (ammonium salt) (16:0 Liss RhodB-PE, λ_Ex/λ_Em = 560/583 nm) from Avanti Polar Lipids (Alabaster, AL, USA). N-isopropylacrylamide (NIPA) as a monomer, N,N′-methylene-bis-acrylamide (MBA) as a cross linker, fluorescein-o-acrylate (FA, λ_Ex/λ_Em = 490/520 nm) as a fluorescent monomer, ammonium persulfate (APS) as an initiator, N,N,N′,N′-tetramethylenediamine (TEMED) as an accelerator, sorbitane monostearate (Span 60) as a surfactant, 2,2-diethoxyacetophenone (DEAP) as a photoinitiator, and solvents (dimethyl sulfoxide, cyclohexane, chloroform, methanol, and ethanol of chemical grade) are purchased from Sigma Aldrich (Saint Louis, MO, USA). Water purified by RiOs-16 Essential Water Purification System (EMD Millipore, Billerica, MA, USA) at the resistivity of 16 MΩ·cm was used in all experiments. All chemicals are used as purchased without further purification.

Isoprene (99%), sec-butyl lithium (1.4 M in cyclohexane), n-butyllithium (2.0 M in cyclohexane), and N,N,N,N-tetramethylenediamine (99.5%) were obtained from Sigma-Aldrich. Dried methanol (99.9%) and divinylbenzene (98%, mixture of isomers), were purchased from Merck (Germany). Butylated hydroxyToluene (BHT), 99% was obtained from Fischer Scientific. Toluene (HPLC grade), benzene (99.9%), Isoprene and divinylbenzene were dried and degassed by several freeze–pump–thaw cycles over calcium hydride (CaH₂) (Aldrich), on a vacuum line. A customised reactor, “Christmas tree” reaction vessel was used to carry out all the syntheses. The reaction vessel was washed thoroughly with solvents and evacuated overnight followed by rinsing with living poly(styryl)lithium solution to react with and remove any trace amount of impurities remaining in the vessel. The apparatus was further evacuated before the polymerisation.