0228cf45a29ea62752cb5712b45edb0d pmc_10674355.pdf d8e26cb05c6c2c0811fbedc925fc6bc4547460e7 pmc_10674355.pdf b470ab2eedf41ff5f0dd651cedf47216cd9099e33d5a2a4f52b9a78e16d692e1 pmc_10674355.pdf Title: Efficient Delivery of Antimicrobial Peptides in an Innovative, Slow-Release Pharmacological Formulation Subject: Both nanostructure and multivalency enhance the biological activities of antimicrobial peptides (AMPs), whose mechanism of action is cooperative. In addition, the efficacy of a particular AMP should benefit from a steady concentration at the local place of action and, therefore, from a slow release after a dynamic repository. In the context of emerging multi-resistant bacterial infections and the urgent need for novel and effective antimicrobial drugs, we tested these concepts through the engineering of four AMPs into supramolecular complexes as pharmacological entities. For that purpose, GWH1, T22, Pt5, and PaD, produced as GFP or human nidogen-based His-tagged fusion proteins, were engineered as self-assembling oligomeric nanoparticles ranging from 10 to 70 nm and further packaged into nanoparticle-leaking submicron granules. Since these materials slowly release functional nanoparticles during their time-sustained unpacking, they are suitable for use as drug depots in vivo. In this context, a particular AMP version (GWH1-NIDO-H6) was selected for in vivo validation in a zebrafish model of a complex bacterial infection. The GWH1-NIDO-H6-secreting protein granules are protective in zebrafish against infection by the multi-resistant bacterium Stenotrophomonas maltophilia, proving the potential of innovative formulations based on nanostructured and slowly released recombinant AMPs in the fight against bacterial infections. Keywords: recombinant proteins; drug delivery; antimicrobial peptide; secretory granules; microparticles Author: Naroa Serna, Hèctor López-Laguna, Patricia Aceituno, Mauricio Rojas-Peña, Eloi Parladé, Eric Voltà-Durán, Carlos Martínez-Torró, Julieta M. Sánchez, Angela Di Somma, Jose Vicente Carratalá, Andrea L. Livieri, Neus Ferrer-Miralles, Esther Vázquez, Ugutz Unzueta, Nerea Roher and Antonio Villaverde Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Mon Nov 20 07:53:45 2023 CET ModDate: Mon Nov 20 07:56:20 2023 CET Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 14 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 1743011 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- ETLENW+VnURWPalladioL-Bold Type 1 Custom yes yes no 10 0 IGDFIY+VnURWPalladioL Type 1 Custom yes yes no 15 0 XSDPME+URWPalladioL-Bold Type 1 Custom yes yes no 19 0 CSFVSM+URWPalladioL-Roma Type 1 Custom yes yes no 24 0 UBXCDJ+URWPalladioL-Ital Type 1 Custom yes yes no 28 0 HHBSSZ+CMSY10 Type 1 Builtin yes yes no 32 0 IAFAOG+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 51 0 MPJNCG+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 89 0 MPJNCH+PalatinoLinotype CID TrueType Identity-H yes yes yes 92 0 MPJNCI+PalatinoLinotype TrueType WinAnsi yes yes no 98 0 MPJNCK+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 101 0 MPJOAF+PalatinoLinotype TrueType MacRoman yes yes no 104 0 MPJOJD+PalatinoLinotype,Italic TrueType MacRoman yes yes no 107 0 OAMRUE+EURM10 Type 1 Builtin yes yes no 114 0 MPJNCG+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 131 0 MPJNCH+PalatinoLinotype CID TrueType Identity-H yes yes yes 134 0 MPJNCI+PalatinoLinotype TrueType WinAnsi yes yes no 140 0 MPJNCK+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 143 0 MPJOAF+PalatinoLinotype TrueType MacRoman yes yes no 146 0 MPJNCG+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 160 0 MPJNCH+PalatinoLinotype CID TrueType Identity-H yes yes yes 163 0 MPJNCI+PalatinoLinotype TrueType WinAnsi yes yes no 169 0 MPJNCK+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 172 0 MPJOAF+PalatinoLinotype TrueType MacRoman yes yes no 175 0 MPJNCG+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 192 0 MPJNCH+PalatinoLinotype CID TrueType Identity-H yes yes yes 195 0 MPJNCI+PalatinoLinotype TrueType WinAnsi yes yes no 201 0 MPJNCK+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 204 0 MPJOAF+PalatinoLinotype TrueType MacRoman yes yes no 207 0 MPUDFL+PazoMath Type 1 Builtin yes yes no 221 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2024-02-02 02:32:36 CET RepresentationInformation: pmc_10674355.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2024-01-09 02:28:49 CET Size: 1743011 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 472 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: section.1 Item: Title: Materials and Methods Destination: section.2 Children: Item: Title: Genetic Design, Production, and Purification Destination: subsection.2.1 Item: Title: Purity, Concentration, and Integrity Destination: subsection.2.2 Item: Title: Size, Ultrastructural Morphometry and Surface Charge Characterization Destination: subsection.2.3 Item: Title: Synthesis of Secretory Granules Destination: subsection.2.4 Item: Title: Chemical Detection of Zinc within Secretory Granules Destination: subsection.2.5 Item: Title: Protein Release from Secretory Granules Destination: subsection.2.6 Item: Title: In Vitro Bactericidal Assay in Staphylococcus aureus Destination: subsection.2.7 Item: Title: Zebrafish Husbandry and Breeding Destination: subsection.2.8 Item: Title: In Vivo Biodistribution in Zebrafish Larvae Destination: subsection.2.9 Item: Title: In Vivo Model of Infection: Stenotrophomonas maltophilia (K279 Strain) in Adult Zebrafish Destination: subsection.2.10 Item: Title: 3D Modelling Destination: subsection.2.11 Item: Title: Statistical Methods Destination: subsection.2.12 Item: Title: Results Destination: section.3 Item: Title: Discussion Destination: section.4 Item: Title: Conclusions Destination: section.5 Item: Title: References Destination: section.A. Info: Title: Efficient Delivery of Antimicrobial Peptides in an Innovative, Slow-Release Pharmacological Formulation Author: Naroa Serna, Hèctor López-Laguna, Patricia Aceituno, Mauricio Rojas-Peña, Eloi Parladé, Eric Voltà-Durán, Carlos Martínez-Torró, Julieta M. Sánchez, Angela Di Somma, Jose Vicente Carratalá, Andrea L. Livieri, Neus Ferrer-Miralles, Esther Vázquez, Ugutz Unzueta, Nerea Roher and Antonio Villaverde Subject: Both nanostructure and multivalency enhance the biological activities of antimicrobial peptides (AMPs), whose mechanism of action is cooperative. In addition, the efficacy of a particular AMP should benefit from a steady concentration at the local place of action and, therefore, from a slow release after a dynamic repository. In the context of emerging multi-resistant bacterial infections and the urgent need for novel and effective antimicrobial drugs, we tested these concepts through the engineering of four AMPs into supramolecular complexes as pharmacological entities. For that purpose, GWH1, T22, Pt5, and PaD, produced as GFP or human nidogen-based His-tagged fusion proteins, were engineered as self-assembling oligomeric nanoparticles ranging from 10 to 70 nm and further packaged into nanoparticle-leaking submicron granules. Since these materials slowly release functional nanoparticles during their time-sustained unpacking, they are suitable for use as drug depots in vivo. In this context, a particular AMP version (GWH1-NIDO-H6) was selected for in vivo validation in a zebrafish model of a complex bacterial infection. The GWH1-NIDO-H6-secreting protein granules are protective in zebrafish against infection by the multi-resistant bacterium Stenotrophomonas maltophilia, proving the potential of innovative formulations based on nanostructured and slowly released recombinant AMPs in the fight against bacterial infections. 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