Poloxamer f127

A two-factor full factorial design was employed to statically analyze the influence of the two polymers (i.e., poloxamer and TPGS) and to obtain an optimized rapamycin polymeric micelle composition using Design-Expert^® software (version 12, Stat-Ease Inc., Minneapolis, MN, USA). Nine formulae were prepared in six replicates, giving a total of 54 runs. Different weight ratios of TPGS were mixed with either poloxamer F127 (BASF, Geismar, LA, USA) or P123 (Sigma-Aldrich, St. Louis, MO, USA). Thus, the independent variables were: poloxamer type (A) and weight ratio of TPGS to total copolymer in nanomicellar mixture (B). The composition of the investigated formulae is shown in Table 1. During the screening studies, polymer content was kept constant at 25 mg∙mL⁻¹. The responses selected to achieve optimized micelle formulation were maximize entrapment efficiency (Y₁), minimize micellar size (Y₂), minimize polydispersity index (Y₃), zeta potential (Y₄), maximize thermodynamic stability (Y₅) and maximize in vitro flux (Y₆), as shown in Table 1. Analysis of variance (ANOVA) was carried out to estimate the significance of the model. Probability p-values (p < 0.05) denoted significance.

Bilirubin (BR), β-CD, γ-PGA (700 kDa), cysteamine (Cys), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), N-hydroxy-succinimide (NHS), morpholine ethanesulfonic acid (MES), and 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Poloxamer F127 was obtained from BASF (Ludwigshafen, Germany). DMEM medium, foetal bovine serum (FBS), bovine serum albumin (BSA), and trypsin were purchased from Gibco (Gibco-BRL, Carlsbad, CA, USA). Haematoxylin and eosin (H&E) staining kit, primary antibodies of tumour necrosis factor α (TNF-α) (ab215188), myeloperoxidase (MPO) (ab208670), CD86 (ab243887), vascular endothelial growth factor (VEGF) (ab32152), transforming growth factor β (TGF-β) (ab215715), and fibroblast growth factor 1 (FGF-1) (ab179455) were purchased from Abcam (Shanghai, China).