c7482b90ca02ccac1971988be8832219 Phar_a2022v14n12p2644iENG.pdf bb88e92562b83d2771f192729ee309f0757bfd1c Phar_a2022v14n12p2644iENG.pdf c08604a9fc6eac3cad8db26bb700f4697e1da19ed4a848110d63d982d7e4134c Phar_a2022v14n12p2644iENG.pdf Title: The Diphtheria Toxin Translocation Domain Impairs Receptor Selectivity in Cancer Cell-Targeted Protein Nanoparticles Subject: Protein-based materials intended as nanostructured drugs or drug carriers are progressively gaining interest in nanomedicine, since their structure, assembly and cellular interactivity can be tailored by recruiting functional domains. The main bottleneck in the development of deliverable protein materials is the lysosomal degradation that follows endosome maturation. This is especially disappointing in the case of receptor-targeted protein constructs, which, while being highly promising and in demand in precision medicines, enter cells via endosomal/lysosomal routes. In the search for suitable protein agents that might promote endosome escape, we have explored the translocation domain (TD) of the diphtheria toxin as a functional domain in CXCR4-targeted oligomeric nanoparticles designed for cancer therapies. The pharmacological interest of such protein materials could be largely enhanced by improving their proteolytic stability. The incorporation of TD into the building blocks enhances the amount of the material detected inside of exposed CXCR4+ cells up to around 25-fold, in absence of cytotoxicity. This rise cannot be accounted for by endosomal escape, since the lysosomal degradation of the new construct decreases only moderately. On the other hand, a significant loss in the specificity of the CXCR4-dependent cellular penetration indicates the unexpected role of the toxin segment as a cell-penetrating peptide in a dose-dependent and receptor-independent fashion. These data reveal that the diphtheria toxin TD displayed on receptor-targeted oligomeric nanoparticles partially abolishes the exquisite receptor specificity of the parental material and it induces nonspecific internalization in mammalian cells. Keywords: recombinant proteins; nanoparticles; diphtheria toxin; self-assembling; cell-penetrating peptide Author: Eric Voltà-Durán, Julieta M. Sánchez, Eloi Parladé, Naroa Serna, Esther Vazquez, Ugutz Unzueta and Antonio Villaverde Creator: LaTeX with hyperref package Producer: pdfTeX-1.40.18 CreationDate: Tue Nov 29 12:13:18 2022 CET ModDate: Tue Nov 29 12:16:03 2022 CET Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 13 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 2487362 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- IYDCBB+VnURWPalladioL Type 1 Custom yes yes no 10 0 FKMBJB+URWPalladioL-Bold Type 1 Custom yes yes no 15 0 JGWZCX+URWPalladioL-Roma Type 1 Custom yes yes no 20 0 PNIARF+URWPalladioL-Ital Type 1 Custom yes yes no 24 0 NCRKTZ+VnURWPalladioL-Bold Type 1 Custom yes yes no 28 0 IAFAOG+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 47 0 OJYQXC+CMSY10 Type 1 Builtin yes yes no 78 0 CAMXTK+EURM10 Type 1 Builtin yes yes no 82 0 BJNNDF+PalatinoLinotype-Bold CID TrueType Identity-H yes yes yes 93 0 BJNNEG+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 99 0 BJNNFH+PalatinoLinotype-Italic CID TrueType Identity-H yes yes yes 105 0 BJNNFI+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 111 0 BJNNFJ+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 114 0 BJNNFK+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 117 0 BJNNDF+PalatinoLinotype-Bold CID TrueType Identity-H yes yes yes 132 0 BJNNEG+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 138 0 BJNNFH+PalatinoLinotype-Italic CID TrueType Identity-H yes yes yes 144 0 BJNNFI+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 150 0 BJNNFJ+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 153 0 BJNNFK+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 156 0 BJNNDF+PalatinoLinotype-Bold CID TrueType Identity-H yes yes yes 179 0 BJNNEG+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 185 0 BJNNFH+PalatinoLinotype-Italic CID TrueType Identity-H yes yes yes 191 0 BJNNFI+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 197 0 BJNNFJ+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 200 0 BJNNFK+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 203 0 BJNNDF+PalatinoLinotype-Bold CID TrueType Identity-H yes yes yes 217 0 BJNNEG+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 223 0 BJNNFH+PalatinoLinotype-Italic CID TrueType Identity-H yes yes yes 229 0 BJNNFI+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 235 0 BJNNFJ+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 238 0 BJNNFK+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 241 0 Jhove (Rel. 1.22.1, 2019-04-17) Date: 2023-03-09 02:36:07 CET RepresentationInformation: Phar_a2022v14n12p2644iENG.pdf ReportingModule: PDF-hul, Rel. 1.12.1 (2019-04-17) LastModified: 2023-03-08 21:29:56 CET Size: 2487362 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 496 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@44c8afef Item: Title: Materials and Methods Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7b69c6ba Children: Item: Title: Genetic Design and Protein Production and Purification Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@46daef40 Item: Title: Three-Dimensional Protein Modelling and Visualization Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@12f41634 Item: Title: Protein Purity, Integrity and Concentration Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@13c27452 Item: Title: Size Distribution and Fluorescence Emission Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@262b2c86 Item: Title: Cell Culture and Confocal Laser Scanning Microscopy Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@371a67ec Item: Title: Cell Cytometry Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@5ed828d Item: Title: Cell Viability Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@50d0686 Item: Title: Proteolytic Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7a3d45bd Item: Title: Statistical Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1e7c7811 Item: Title: Results Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77ec78b9 Item: Title: Discussion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1a3869f4 Item: Title: Conclusions Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@a38d7a3 Item: Title: References Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77f99a05 Info: Title: The Diphtheria Toxin Translocation Domain Impairs Receptor Selectivity in Cancer Cell-Targeted Protein Nanoparticles Author: Eric Voltà-Durán, Julieta M. Sánchez, Eloi Parladé, Naroa Serna, Esther Vazquez, Ugutz Unzueta and Antonio Villaverde Subject: Protein-based materials intended as nanostructured drugs or drug carriers are progressively gaining interest in nanomedicine, since their structure, assembly and cellular interactivity can be tailored by recruiting functional domains. The main bottleneck in the development of deliverable protein materials is the lysosomal degradation that follows endosome maturation. This is especially disappointing in the case of receptor-targeted protein constructs, which, while being highly promising and in demand in precision medicines, enter cells via endosomal/lysosomal routes. In the search for suitable protein agents that might promote endosome escape, we have explored the translocation domain (TD) of the diphtheria toxin as a functional domain in CXCR4-targeted oligomeric nanoparticles designed for cancer therapies. The pharmacological interest of such protein materials could be largely enhanced by improving their proteolytic stability. The incorporation of TD into the building blocks enhances the amount of the material detected inside of exposed CXCR4+ cells up to around 25-fold, in absence of cytotoxicity. This rise cannot be accounted for by endosomal escape, since the lysosomal degradation of the new construct decreases only moderately. On the other hand, a significant loss in the specificity of the CXCR4-dependent cellular penetration indicates the unexpected role of the toxin segment as a cell-penetrating peptide in a dose-dependent and receptor-independent fashion. These data reveal that the diphtheria toxin TD displayed on receptor-targeted oligomeric nanoparticles partially abolishes the exquisite receptor specificity of the parental material and it induces nonspecific internalization in mammalian cells. 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