Fig 1: In vivo blood–brain barrier (BBB) penetration.Notes: Representative fluorescence photomicrographs of immunohistochemistry on brain sections, of the specified area (marked with orange box) using a 6× Histag antibody to detect the TIMP-1-Histag fusion protein. The nucleus was labeled with DAPI. The TIMP-1 NPs + Ps80 group shows labeling indicating BBB penetration of TIMP-1 NPs + Ps80 as indicated by white arrows.Abbreviations: TIMP, tissue inhibitor of matrix metalloproteinases; DAPI, 4′,6-diamidino-2-phenylindole; NPs, nanoparticles; Ps80, polysorbate 80; PBS, phosphate-buffered saline.
Fig 2: TIMP-1 release and stability: release of TIMP-1 from optimal PLGA3 NPs under in vitro conditions. Inset is representative Western blot of the protein released from the NPs, suggesting that release of TIMP-1 is stable after 48 hours. Data are means ± standard error of mean; n=3.Abbreviations: NPs, nanoparticles; TIMP, tissue inhibitor of matrix metalloproteinases; PLGA, poly(lactic-co-glycolic acid); Std, standard.
Fig 3: (A–C) Toxicity studies. (A) LY assay on RBCEC, showing no significant impact of NPs on endothelial cell permeability for LY compared to control (no NPs), indicating that NPs caused no significant opening of the endothelial cell monolayer. Ps80 alone and TIMP-1 alone were used as controls. (B) LDH assay, also showing that NPs were not toxic to the RBCEC compared to control. Ps80 alone and TIMP-1 alone were used as controls. (C) Representative fluorescence photomicrographs of ZO1 immunocytochemistry on RBCEC treated with TIMP-1 NPs and TIMP-1 NPs + Ps80, indicating no significant difference between the groups.Note: Values represent means ± standard error of mean of three independent experiments.Abbreviations: LY, Lucifer yellow; RBCEC, primary rat brain capillary endothelial cells; NPs, nanoparticles; TIMP, tissue inhibitor of matrix metalloproteinases; Ps80, polysorbate 80; LDH, lactate dehydrogenase; LY, Lucifer yellow.
Fig 4: Graphical Summary.Notes: 1. In this study, we formulated TIMP-1 loaded NPs using multiple emulsion solvent evaporation method. The NPs were coated with Ps80 to improve their BBB penetration. 2. We used RBE4/RBCEC as our in vitro BBB model. 3. We compared toxicity of Ps80 coated and non-coated NPs using LY assay, LDH assay, and ZO1 immuno. And we have shown that either of the NPs are nontoxic to endothelial cells. 4. Next, we evaluated the BBB penetration for dye NPs using spectrophotometry and ELISA for TIMP-1 NPs and we have shown that Ps80 coated enhances the in vitro BBB penetration. 5. Finally, we evaluated in vivo BBB penetration by injecting the Ps80 coated TIMP-1 loaded NPs through tail vein injection and it showed that BBB penetration of intravenously injected TIMP-1 NPs + Ps80. 6. To summarize, we have developed TIMP-1 loaded PLGA NPs which can deliver TIMP-1 in a sustained release manner and can cross the BBB. the in vitro and in vivo results have shown that NPs are nontoxic to endothelial cells and they have BBB penetration.Abbreviations: NPs, nanoparticles; TIMP, tissue inhibitor of matrix metalloproteinases; Ps80, polysorbate 80; LDH, lactate dehydrogenase; LY, Lucifer yellow; BBB, blood-brain barrier; ELISA, enzyme-linked immunosorbent assay; immuno, immunocytochemistry; RBE4, rat brain endothelial cell line; RBCEC, rat brain capillary endothelial cells; PLGA, Poly(lactic-co-glycolic acid); ZO1, zona occludens 1.
Fig 5: Formulation optimization.Abbreviations: TIMP, tissue inhibitor of matrix metalloproteinases; PLGA, poly(lactic-co-glycolic acid); PDI, polydispersity index.
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