Journal: International journal of biological macromolecules

Article Title: In vitro and in vivo characterization of human serum albumin-based PEGylated nanoparticles for BDNF and NT3 codelivery.

doi: 10.1016/j.ijbiomac.2024.130726

Figure Lengend Snippet: Fig. 1. Physicochemical characterization of PEGylated HSA-NT3-BDNF (NT3 10 mg L−1, BDNF 10 mg L−1) nanoparticles. The particle size distribution was measured by intensity (A) and by number (B), the nanoparticle concentration (C), and the zeta potential (D) were measured against Ringer buffer at pH 4.9 and 0.15 M ionic strength. The intensity (percent) indicates the size frequency of the most common particles. All syntheses were performed fifteen times, and the error bars represent the mean ± standard deviation (SD). The individual curves indicate the distribution of NP diameters from 24 samples.

Article Snippet: Unfiltered stock solutions (typically 250 mg L− 1) of carrier-free recombinant human BDNF (rhBDNF) (248-N4-250/CF; R&D Systems, Canada), as well as carrier-free recombinant human NT3 (rhNT3) (248- BDB-250/CF; R&D Systems), were prepared by dissolving lyophilizates of known concentrations in phosphate-buffered saline (PBS) (pH 7.4 ± 0.2, 0.15 M; Biomed, Lublin, Poland) and storing them for no longer than 2 months at − 20 ◦C.

Techniques: Concentration Assay, Zeta Potential Analyzer, Standard Deviation

Journal: International journal of biological macromolecules

Article Title: In vitro and in vivo characterization of human serum albumin-based PEGylated nanoparticles for BDNF and NT3 codelivery.

doi: 10.1016/j.ijbiomac.2024.130726

Figure Lengend Snippet: Fig. 2. Stability of PEGylated HSA-NT3-BDNF (NT 10 mg L−1) nanoparticles over 90 days after formulation. The particle size distribution was measured by intensity (A) and by number (B), the zeta potential was measured against Ringer buffer (C), and the loading of NTs was measured via ELISA (D). The intensity (percent) indicates the size frequency of the most common particles. All syntheses were performed six times, and the error bars represent the mean ± standard deviation (SD). The individual curves indicate the distribution of NP diameters from 8 samples. Note that the dispersed type of PEGylated HSA-NT3-BDNF nanoparticles was maintained for 90 days.

Article Snippet: Unfiltered stock solutions (typically 250 mg L− 1) of carrier-free recombinant human BDNF (rhBDNF) (248-N4-250/CF; R&D Systems, Canada), as well as carrier-free recombinant human NT3 (rhNT3) (248- BDB-250/CF; R&D Systems), were prepared by dissolving lyophilizates of known concentrations in phosphate-buffered saline (PBS) (pH 7.4 ± 0.2, 0.15 M; Biomed, Lublin, Poland) and storing them for no longer than 2 months at − 20 ◦C.

Techniques: Formulation, Zeta Potential Analyzer, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: International journal of biological macromolecules

Article Title: In vitro and in vivo characterization of human serum albumin-based PEGylated nanoparticles for BDNF and NT3 codelivery.

doi: 10.1016/j.ijbiomac.2024.130726

Figure Lengend Snippet: Fig. 4. Release of NT3 (in black, circle dots) and BDNF (in gray, square) from the PEGylated HSA-NT3-BDNF nanoparticles in a cell-free system. Kinetics of neurotrophin release over 2 months. n = 3; statistics: Two-way ANOVA with Bonferroni post hoc correction was used to compare the time course between NT3 and BDNF. The relative release of the adsorbed neurotrophins was calcu lated via ELISA.

Article Snippet: Unfiltered stock solutions (typically 250 mg L− 1) of carrier-free recombinant human BDNF (rhBDNF) (248-N4-250/CF; R&D Systems, Canada), as well as carrier-free recombinant human NT3 (rhNT3) (248- BDB-250/CF; R&D Systems), were prepared by dissolving lyophilizates of known concentrations in phosphate-buffered saline (PBS) (pH 7.4 ± 0.2, 0.15 M; Biomed, Lublin, Poland) and storing them for no longer than 2 months at − 20 ◦C.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: International journal of biological macromolecules

Article Title: In vitro and in vivo characterization of human serum albumin-based PEGylated nanoparticles for BDNF and NT3 codelivery.

doi: 10.1016/j.ijbiomac.2024.130726

Figure Lengend Snippet: Fig. 3. Representative binding curves for the interaction between HSA and BDNF (upper curve, Part A) and NT3; (upper curve, Part B). Representative MST/TRIC time traces of 40 nM labeled BDNF (lower curve, Part A) and 20 nM labeled NT3; (lower curve, Part B) in PBS supplemented with 1 mM HSA for the NT3 assay and 0.5 mM HSA for BDNF. Cold (blue) and hot (red) regions were defined for central data analysis to calculate Fnorm. The data for both figures are from instrument N06. Diagrams exported from MOAA. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Unfiltered stock solutions (typically 250 mg L− 1) of carrier-free recombinant human BDNF (rhBDNF) (248-N4-250/CF; R&D Systems, Canada), as well as carrier-free recombinant human NT3 (rhNT3) (248- BDB-250/CF; R&D Systems), were prepared by dissolving lyophilizates of known concentrations in phosphate-buffered saline (PBS) (pH 7.4 ± 0.2, 0.15 M; Biomed, Lublin, Poland) and storing them for no longer than 2 months at − 20 ◦C.

Techniques: Binding Assay, Labeling

Journal: International journal of biological macromolecules

Article Title: In vitro and in vivo characterization of human serum albumin-based PEGylated nanoparticles for BDNF and NT3 codelivery.

doi: 10.1016/j.ijbiomac.2024.130726

Figure Lengend Snippet: Fig. 6. Release of NT3 (in black) and BDNF (in gray) from the PEGylated HSA-NT3-BDNF nanoparticles in vitro. Kinetics of NT3 and BDNF release in culture media supplemented with (A) the complex (10 % FBS) or (B) serum starved (1 % FBS). The time course was >72 h. (n = 3; statistics: Two-way ANOVA with Bonferroni post hoc correction to compare the time course and either complex media or serum starvation conditions). The relative release of the adsorbed neurotrophins was calculated via ELISA.

Article Snippet: Unfiltered stock solutions (typically 250 mg L− 1) of carrier-free recombinant human BDNF (rhBDNF) (248-N4-250/CF; R&D Systems, Canada), as well as carrier-free recombinant human NT3 (rhNT3) (248- BDB-250/CF; R&D Systems), were prepared by dissolving lyophilizates of known concentrations in phosphate-buffered saline (PBS) (pH 7.4 ± 0.2, 0.15 M; Biomed, Lublin, Poland) and storing them for no longer than 2 months at − 20 ◦C.

Techniques: In Vitro, Enzyme-linked Immunosorbent Assay

Journal: International journal of biological macromolecules

Article Title: In vitro and in vivo characterization of human serum albumin-based PEGylated nanoparticles for BDNF and NT3 codelivery.

doi: 10.1016/j.ijbiomac.2024.130726

Figure Lengend Snippet: Fig. 7. Intracellular levels of BDNF and NT3 in the ARPE-19 cell line after 72 h of incubation with PEGylated HSA-NT3-BDNF.

Article Snippet: Unfiltered stock solutions (typically 250 mg L− 1) of carrier-free recombinant human BDNF (rhBDNF) (248-N4-250/CF; R&D Systems, Canada), as well as carrier-free recombinant human NT3 (rhNT3) (248- BDB-250/CF; R&D Systems), were prepared by dissolving lyophilizates of known concentrations in phosphate-buffered saline (PBS) (pH 7.4 ± 0.2, 0.15 M; Biomed, Lublin, Poland) and storing them for no longer than 2 months at − 20 ◦C.

Techniques: Incubation

Journal: International journal of biological macromolecules

Article Title: In vitro and in vivo characterization of human serum albumin-based PEGylated nanoparticles for BDNF and NT3 codelivery.

doi: 10.1016/j.ijbiomac.2024.130726

Figure Lengend Snippet: Fig. 5. The influence of the neurotrophin concentrations in PEGylated HSA-NT3-BDNF nanoparticles on viability and proliferation (in relation to the untreated control). After 24 h of preincubation, ARPE-19 (A) or L-929 (B) cells were treated with 5, 10 or 20 mg L−1 neurotrophin in PEGylated HSA-NT3-BDNF (described in the picture as HSA-NT3-BDNF-PEG) nanoparticles and stored in regular cell culture conditions. After 48 h, alamarBlue™ reagent was added to the cell culture mixture according to the manufacturer's instructions. The experiment was performed in three independent replicates. One-way ANOVA (GraphPad Prism) did not reveal any significant differences.

Article Snippet: Unfiltered stock solutions (typically 250 mg L− 1) of carrier-free recombinant human BDNF (rhBDNF) (248-N4-250/CF; R&D Systems, Canada), as well as carrier-free recombinant human NT3 (rhNT3) (248- BDB-250/CF; R&D Systems), were prepared by dissolving lyophilizates of known concentrations in phosphate-buffered saline (PBS) (pH 7.4 ± 0.2, 0.15 M; Biomed, Lublin, Poland) and storing them for no longer than 2 months at − 20 ◦C.

Techniques: Control, Cell Culture

Journal: International journal of biological macromolecules

Article Title: In vitro and in vivo characterization of human serum albumin-based PEGylated nanoparticles for BDNF and NT3 codelivery.

doi: 10.1016/j.ijbiomac.2024.130726

Figure Lengend Snippet: Fig. 8. NT3 and BDNF release profiles in the retina and vitreous body of BALB/c mice.

Article Snippet: Unfiltered stock solutions (typically 250 mg L− 1) of carrier-free recombinant human BDNF (rhBDNF) (248-N4-250/CF; R&D Systems, Canada), as well as carrier-free recombinant human NT3 (rhNT3) (248- BDB-250/CF; R&D Systems), were prepared by dissolving lyophilizates of known concentrations in phosphate-buffered saline (PBS) (pH 7.4 ± 0.2, 0.15 M; Biomed, Lublin, Poland) and storing them for no longer than 2 months at − 20 ◦C.

Techniques:

Journal: The Journal of Neuroscience

Article Title: A Changing Pattern of Brain-Derived Neurotrophic Factor Expression Correlates with the Rearrangement of Fibers during Cochlear Development of Rats and Mice

doi: 10.1523/JNEUROSCI.19-08-03033.1999

Figure Lengend Snippet: Characterization of anti-BDNF antibody.A, Cross-reactivity of anti-BDNF antibody with neurotrophins NT-3, NT-4, and NGF was tested in Western blot analysis. Anti-BDNF recognized only rhBDNF but not rhNT-3, rhNT-4, or rhNGF.B, Sensitivity of anti-BDNF antibody was tested in Western blot using indicated concentrations of BDNF. The minimal amount rhBDNF recognized by anti-BDNF antibody was 5 ng. C, In a Western blot with rat cochlear protein, anti-BDNF antibody recognized a protein (arrow) close to the molecular weight of rhBDNF, which is likely to be the mature BDNF protein. Anti-BDNF antibody also recognized proteins of ∼20 and ∼30 kDa, probably BDNF precursor proteins. D, Anti-BDNF immunoreaction (Anti-BDNF) in hair cells (arrowheads and vertical arrows) was completely abolished by preabsorption of the antibody with rhBDNF (Anti-BDNF+rhBDNF) but not with rhNT-3 (Anti-BDNF+rhNT-3). Immunoreaction in the tectorial membrane (TM) was nonspecific. Scale bar, 20 μm.

Article Snippet: For neutralization of BDNF immunoreaction, the anti-BDNF antibody was preincubated with either recombinant human BDNF (rhBDNF) (100 ng/ml; Regeneron Pharmaceuticals, Tarrytown, NY) or recombinant human NT-3 (rhNT-3) (100 ng/ml; Regeneron Pharmaceuticals) before immunohistochemical staining.

Techniques: Western Blot, Molecular Weight

Journal: The Journal of Neuroscience

Article Title: A Changing Pattern of Brain-Derived Neurotrophic Factor Expression Correlates with the Rearrangement of Fibers during Cochlear Development of Rats and Mice

doi: 10.1523/JNEUROSCI.19-08-03033.1999

Figure Lengend Snippet: Localization of BDNF mRNA in the organ of Corti and the spiral ganglion of rats by in situhybridization. Although no BDNF mRNA was detected at P3, BDNF mRNA was localized in OHCs (vertical arrows), pillar cells (PC), Deiters’ cells (DC), and border cells (BC) of the organ of Corti at P6. At P8, BDNF mRNA was noted restrictively in Deiters’ and border cells. In the spiral ganglion (SG), BDNF hybridization signal in neurons was not effected by cohybridization with an excess of nonlabeled NT-3 antisense (BDNF+nlNT-3) but was completely abolished by cohybridization with an excess of nonlabeled BDNF antisense (BDNF+nlBDNF). Scale bars, 20 μm.

Article Snippet: For neutralization of BDNF immunoreaction, the anti-BDNF antibody was preincubated with either recombinant human BDNF (rhBDNF) (100 ng/ml; Regeneron Pharmaceuticals, Tarrytown, NY) or recombinant human NT-3 (rhNT-3) (100 ng/ml; Regeneron Pharmaceuticals) before immunohistochemical staining.

Techniques: Hybridization