CB is thankful to Fondazione Umano Progresso for fellowship. Footnotes Author contributions All writers contributed toward data analysis, drafting and revising PCI-24781 (Abexinostat) the paper and consent to end up being in charge of all areas of the ongoing function. Disclosure The authors report no conflicts appealing within this ongoing work.. (117K) GUID:?4736D1B9-B146-466A-8983-6C404C665B70 Figure S4: In vitro discharge test.Be aware: (A) TZ and (B) DOXO cumulative discharge in vitro. Abbreviations: TZ, trastuzumab; DOXO, doxorubicin. ijn-13-957s4.tif (105K) GUID:?1D81B73F-D3C9-4E87-ACB4-5F3F2A899008 Figure S5: Evaluation of TZ primary structure integrity after release.Take note: SDS-PAGE performed on RTZ, TZ extracted with NaOH (TZ_NaOH) and unloaded TZ (TZ_clean) compared to CTZ as reference. Abbreviations: SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TZ, trastuzumab; RTZ, released TZ; Rabbit polyclonal to EGFP Tag CTZ, control TZ. ijn-13-957s5.tif (674K) GUID:?4370C09F-8E30-4FE1-AC33-C78B0A628A25 Figure S6: Binding of TZ-FITC.Notes: (A) Basal binding of CTZ, RTZ and RIgG labeled with FITC to SKBR3 cell line. (B) Percentage of TZ-FITC binding on SKBR3 after 24 and 48 h of treatment with 2 g mL?1 and 20 g mL?1 of CTZ, RTZ and RIgG. ****P<0.01 vs UNTR. Abbreviations: TZ, trastuzumab; FITC, fluorescein isothiocyanate; CTZ, control TZ; RTZ, released TZ; RIgG, released IgG; UNTR, cells coated with TZ-FITC. ijn-13-957s6.tif (283K) GUID:?E7346F02-CFAD-4BD5-BEBC-C05E37909476 Physique S7: Western blot of unspecific IgG.Notes: Analysis of HER2 expression on SKBR3 cells after treatment with CIgG, RIgG and IgG@PLGA at 2 g mL?1 for 24 h. Values were calculated as ratio between HER2/-actin and normalized with untreated cells (UNTR). Abbreviations: HER2, human epidermal growth factor receptor 2; CIgG, control IgG; RIgG, released IgG; IgG@PLGA, IgG-loaded PLGA NPs; UNTR, cells without treatment. ijn-13-957s7.tif (148K) GUID:?C57EAE34-7961-43E4-ACED-FBF9545F0A49 Figure S8: Confocal microscopy.Notes: Confocal microscopy untreated cells labeled with DAPI (nuclei), EEA1 (early endosomes) and CatD (lysosomes). Scale bar =50 m. Abbreviations: DAPI, 4,6-diamidin-2-fenilindolo; EEA1, Early Endosome Antigen 1; CatD, Cathepsin D. ijn-13-957s8.tif (644K) GUID:?F61735D0-BAFA-4B7E-948E-1C21EDAD107D Physique S9: Western blot of unspecific IgG.Notes: Analysis of pHER2 expression on SKBR3 cells after treatment with TZ@PLGA at 2 g mL?1 for 4 h and 24 h. Values were calculated as ratio between pHER2/HER2 and normalized with untreated cells (UNTR). Abbreviations: HER2, human epidermal growth factor receptor 2; TZ@PLGA, trastuzumab-loaded poly(lactic-co-glycolic) acid nanoparticles; UNTR, cells without treatment. ijn-13-957s9.tif (153K) GUID:?B092A056-60EF-4255-A3EA-EA2B3ED50DF1 Abstract Background We report the development of an efficient antibody delivery system for the incorporation of trastuzumab (TZ) into poly(lactic-co-glycolic) acid nanoparticles (PLGA NPs). The aim of the work was to overcome the current limitations in the clinical use of therapeutic antibodies, including immunogenicity, poor pharmacokinetics, low tumor penetration and safety issues. Materials and methods Trastuzumab-loaded PLGA NPs (PLGA-TZ) were synthesized according to a double emulsion method. The same protocol was used to produce control batches of nonspecific IgG-loaded NPs and empty PLGA NPs. After release of TZ from PLGA NPs, the effects on the main biological activities of the antibody were evaluated on SKBR3 (human epidermal growth factor receptor 2 [HER2]-positive breast cancer cell line), including specific binding to HER2, phosphorylation of HER2 (Y1248), degradation of HER2 protein and antibody-dependent cell-mediated cytotoxicity (ADCC) mechanism. In addition, an PCI-24781 (Abexinostat) MTT assay was performed for treating SKBR3 cells with PLGA NPs loaded with TZ and doxorubicin to evaluate the cytotoxic activity of the combined treatment. Results and discussion TZ was gradually released in a prolonged way over 30 days. The physical characterization performed with circular dichroism, Fourier transform infrared and fluorescence spectroscopy PCI-24781 (Abexinostat) of TZ after release demonstrated that no structural alterations occurred compared to the native antibody. In vitro experiments using SKBR3 cells showed that TZ released from PLGA NPs maintained the same biological activity of native TZ. PLGA NPs allowed a good co-encapsulation efficiency of TZ and doxorubicin resulting.