Fluorescein isothiocyanate (fitc)

1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-1000] (DSPE-PEG_1k), 1,2-dihexadecanoyl-rac-glycero-3-phosphocholine (DPPC) and cholesterol were purchased from Avanti Polar Lipids Inc. (Birmingham, AL, USA), IR1048 dye was bought from Sigma-Aldrich (Darmstadt, Germany), while a membrane protein extraction kit was ordered from Beyotime Biotechnology. In addition, 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate (DiI) was ordered from YEASEN Biotechnology (Shanghai) Co., Ltd. (Shanghai, China). The utilized consumables including radio-immunoprecipitation assay (RIPA) lysis buffer, phenylmethanesulfonyl fluoride (PMSF), phosphatase inhibitors, 3,3′-dioctadecyloxacarbocyanine perchlorate (DiO) and enhanced cell counting Kit-8 (CCK-8) were purchased from Beyotime Biotechnology. Further, hoechst 33342 was purchased from US EVERBRIGHT, Suzhou, China. Fluorescein isothiocyanate (FITC) and Rhodamine B were ordered from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Trypsin was bought from Gibco (Waltham, MA, USA). Calcein-AM and Propidium Iodide were ordered from Invitrogen (Waltham, MA, USA). In addition, all the chemicals used were not further purified. What is more, pure water used here was Milli-Q of 18.25 MΩ·cm (23 °C, EMD Millipore, Burlington, MA, USA).

Polyamidoamine dendrimers, generation 5, primary amine surface, and methanol solution (PAMAM G5.NH₂) was purchased from Dendritech (Midland, Michigan, USA). The heterofunctional PEG polymer with a methoxy and succinimidyl ester functional groups (mPEG-NHS, Mw: 2000), fluorescein isothiocyanate (FITC), Phosphate buffer (pH 7.4), Borate buffer (pH 8.4), paeonol (PAE, >99%), and Hydroxypropyl methyl cellulose (HPMC, 30000 mPa·s) were purchased from Shanghai Yuanye Biological Technology Co., Ltd. (Shanghai, China). All other chemicals were purchased from Shanghai Yuanye Biological Technology Co., Ltd. (Shanghai, China), unless otherwise stated. Methylene Blue and Vitamin E were purchased from Solarbio (Beijing, China). Centrifugal filters (MW = 3000 Da and MW = 10 KDa) and dialysis bag (MW = 3500 Da) were purchased from Amicon (Darmstadt, Germany). (4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazo -lium bromide (MTT) and Deacetylated gellan gum (DGG) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Wistar rats (200–220 g) were purchased from the Wuhan Sericebio Co., Ltd. (Wuhan, China) and were handled according to the Principles of Laboratory Animal Care. The protocols were approved by the Wuhan Sericebio Co., Ltd. (Wuhan, China). Animal Ethical Committee (NO.42000600030537).

Dexamethasone and FITC were purchased from Yuanye Biotechnology (Shanghai, China). Bevacizumab (molecular weight (MW) 149 kDa) was donated by Luye Pharmaceutic (Yantai, China). PLGA 503H was purchased from Evonic Industries (Birmingham, AL, Germany). cRGD-PEG-PLGA was purchased from Xi’an Ruixi biotechnology (Xi’an, China). Micro bicinchoninic acid (BCA) protein assay kits were purchased from Thermo Scientific (Waltham, MA, USA). PEI (MW 25 kDa, branched) and polyvinyl alcohol (PVA) (MW 13–23 kDa) were purchased from Sigma-Aldrich (St Louis, MO, USA). Rhod B was purchased from Sinopharm chemical reagents (Beijing, China). Acetonitrile was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Rabbit VEGF enzyme-linked immunosorbent assay (ELISA) kits were obtained from Elabscience Biotechnology (Wuhan, China). All other chemicals used were of analytical grade and used without further purification.

E. tarda was cultured to logarithmic phase and resuspended in 100 μg/ml FITC (YuanYe Bio-Technology, Shanghai, China), followed by incubation at room temperature for 3 h. The bacteria were collected and washed five times with PBS. The bacteria were resuspended in L-15 medium to 1×10⁸ CFU/ml and incubated with or without (control) 1 μM rPoCXCL10, rPoCXCL10M, or rSumo at room temperature for 2 h. Flounder PBLs were mixed with untreated E. tarda or E. tarda treated with rPoCXCL10, rPoCXCL10M, or rSumo at a multiplicity of infection (MOI) of 1:2. After incubation at 22°C for 2 h, the PBLs were centrifuged and washed with PBS. Trypan blue (0.125% in PBS) was added to the cells to quench the extracellular fluorescence. The cells were washed and suspended in PBS. The intracellular fluorescence signal of the bacteria was determined using a FACScan flow cytometer (BD Biosciences, USA).

In order to label all blood-circulating vessels, an intravascular perfusion of fluorescent tomato lectin was performed [11 (link)]. Anesthetized SD rats were intravenously injected with 100 μl fluorescein isothiocyanate-conjugated tomato lectin (1 mg/ml; Sigma-Aldrich; Merck Millipore) and 500 μl fluorescein isothiocyanate (1 mg/ml; Yuanye Bio-Technology; Shanghai; China). Tomato lectin bound uniformly to the luminal surface of endothelial cells [12 (link)] and labeled all blood vessels with adequate blood supply. At 15 mins after injection, rats were perfused with stroke-physiological saline solution for 5 mins through the left ventricle at pressures of 80-120 mmHg under anesthesia. The vitreous humor and retina were isolated from the eyes under an ×2.5 anatomic microscope. The vitreous humor was isolated for VEGF-A ELISA and the retina for hematoxylin and eosin (H&E) staining, periodic acid-Schiff (PAS) staining, fluorescence imaging techniques, and expression of proinflammatory proteins on the 8^th day, at the 4^th week, 6^th week, 8^th week, and 10^th week after induction of DM, respectively.