Formamide

PSF with an average molecular weight of 16 000 was purchased from Aldrich Co., Ltd, China. Chloroacetic chloride (CAC, purity: greater than or equal to 98%) was obtained from Former Derivatives Technology Co., Ltd, China. Anhydrous aluminium chloride with a purity greater than 99% and CS (average viscosity: 100–200 mPa s) with deacetylation exceeding 95% were provided by Aladdin Reagent Co., Ltd, China. Dichloromethane (DCM), ethylenediamine (EDA), carbodiimide (DCC), hydrochloric acid, 25% of glutaraldehyde solution, citric acid (CA), acetic acid and formamide were all supplied by Sinopharm Chemical Reagent Co., Ltd, China. Chlorosulfonic acid was bought by Mars Fine Chemicals Co., Ltd, China. N, N-dimethylacetamide (DMAC) was used as the solvent to cast a film, and absolute ethanol was obtained from Fuyu Fine Chemical Co., Ltd, China. MD44 dialysis bags (average diameter: 28 mm) with the retention range from 8000 to 12 000 were bought from Leibusi company, China. All chemical reagents in this experiment were analytical pure.

The mice were injected intravenously with 50 mg kg⁻¹ Evans Blue solution (Sigma, St. Louis, Missouri, USA), which was left to circulate in vivo for 2 h. The mice were then euthanized and lung tissues were collected. Lung tissues were weighed, cut, and transferred to tubes. Formamide (Sinopharm, Beijing, China) was incubated with lung tissues in an oven to extract Evans Blue. The supernatant was obtained by centrifugation and the absorbance at 620 nm was measured using an enzyme meter.

Silver nitrate (AgNO₃, AR, ≥99.8%), formamide (≥99.5%), DMF (≥99.5%), ethanol and sodium hydroxide (NaOH, AR, ≥96%) were purchased from Sinopharm Chemical Reagent Co. Ltd. n-Pentanol (≥99%) was obtained from Shanghai Lingfeng Chemical Reagent Co. Ltd. Polyvinylpyrrolidone (PVP, M_n = 30 000 g mol⁻¹, pH = 3.64) was obtained from Shanghai Yeasen Biotechnology Co. Ltd. Water was distilled and deionized using a Millipore Milli-Q Purification System and has a resistivity of more than 18.2 MΩ cm. All reagents were used without further purification.

The Miles assay was performed on 8-week-old female C57BL/6J mice to evaluate the effects of SLY on vascular permeability. C57BL/6J mice were purchased from the Animal Care Center of the Academy of Military Medical Science (AMMS; Beijing, China) and housed in a clean room with unlimited access to food and water. TLR4 knockout mice C57BL/10ScNJNju and control mice were obtained from the Model Animal Research Center of Nanjing University. The mice were randomly divided into four groups and intradermally injected in the abdomen with 100 μL THB, 05ZYH33-supernatant, Δsly-supernatant, or CΔsly-supernatant. Four hours after the injection, 100 μL Evans blue dye solution (2.5%, Ourchem, Sinopharm) was injected via the tail vein. The mice were sacrificed by cervical dislocation 30 min after the Evans blue injection, and equal areas (20 mm × 20 mm) of skin surrounding the intradermal injection site were removed from each mouse and completely dried in an oven. The Evans blue dye was eluted from the oven-dried skin into 1 mL formamide (Sinopharm) at 55°C for 2 days, and quantified by spectrophotometry at 630 nm.

RTF solutions were prepared by mixing triethanolamine (Sinopharm Chemical Reagent Co., Ltd, China), formamide (Sinopharm Chemical Reagent Co., Ltd, China), and distilled water by volume. A 30%TEA/40%F/30%W mixture refers to 30% triethanolamine (TEA), 40% formamide (F), 30% water (W) solution, termed as RTF-R1. A 60%TEA/25%F/15%W mixture refers to 60% triethanolamine (TEA), 25% formamide (F), 15% water (W) solution, termed as RTF-R2. A 70%TEA/15%F/15%W refers to 70% triethanolamine (TEA), 15% formamide (F), 15% water (W) solution, termed as RTF-R3.

Chitosan (MW = 200 kDa) was prepared by our laboratory, ovalbumin (OVA) was bought from Sigma-Aldrich (Saint Louis, MO, USA), myoglobin (Mb) and bovine serum albumin (BSA) were bought from Solarbio, N, N-dimethylformamide, formamide, N-methyl-2-pyrrolidone, chlorosulfonic acid, phthalic anhydride, and ethylene glycol came from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). DMEM medium and penicillin–streptomycin came from HyClone (Logan, Utah, USA). Fetal bovine serum (FBS) was acquired from Gibco in Australia; PrimeScript RT reagent Kit (including gDNA Eraser) and SYBR Premix Ex Taq II (Tli RNase H Plus) were bought from Takla in Shiga, Japan. CCK-8 was supplied by ApexBio (Houston, TX, USA), E.Z.N.A. Total RNA Kit was bought by OMEGA Biotechnology Company in, Norcross, GA, USA; ToxinSensor™ Chromogenic LAL Endotoxin Assay Kit was bought from GenScript (Piscatway, NJ, USA).