f5e788a936de7d5c73b16385a1327e3b pmc_10531859.pdf 3bd4948e3c8d83a699064b6277616557ee16c96f pmc_10531859.pdf 1948c11ae8ddccf54e43967f73eae9636148af2d0e1c6f5594919e59e9904e38 pmc_10531859.pdf Title: Role of the Lactide:Glycolide Ratio in PLGA Nanoparticle Stability and Release under Lysosomal Conditions for Enzyme Replacement Therapy of Lysosomal Storage Disorders Subject: Prior studies demonstrated that encapsulation in poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) enhanced the delivery of enzymes used for replacement therapy (ERT) of lysosomal storage disorders (LSDs). This study examined how the copolymer lactide:glycolide ratio impacts encapsulation, physicochemical characteristics, stability, and release under lysosomal conditions. Hyaluronidase, deficient in mucopolysaccharidosis IX, was encapsulated in NPs synthesized using 50:50, 60:40, or 75:25 lactide:glycolide copolymers. All NPs had diameters compatible with cellular transport (168 nm) and polydispersity indexes (0.16) and -potentials (-35 mV) compatible with colloidal stability. Yet, their encapsulation efficiency varied, with 75:25 NPs and 60:40 NPs having the lowest and highest EE, respectively (15% vs. 28%). Under lysosomal conditions, the 50:50 copolymer degraded fastest (41% in 1 week), as expected, and the presence of a targeting antibody coat did not alter this result. Additionally, 60:40 NPs destabilized fastest (<1 week) because of their smaller diameter, and 75:25 NPs did not destabilize in 4 weeks. All formulations presented burst release under lysosomal conditions (56–78% of the original load within 30 min), with 50:50 and 60:40 NPs releasing an additional small fraction after week 1. This provided 4 weeks of sustained catalytic activity, sufficient to fully degrade a substrate. Altogether, the 60:40 NP formulation is preferred given its higher EE, and 50:50 NPs represent a valid alternative, while the highest stability of 75:25 NPs may impair lysosomes. These results can guide future studies aiming to translate PLGA NP-based ERT for this and other LSDs. Keywords: lysosomal storage disorder; enzyme replacement therapy; hyaluronidase; poly(lactide-co-glycolide) nanoparticles; copolymer ratio; nanoparticle stability; enzyme release Author: Maria del Moral, Maximilian Loeck, Eameema Muntimadugu, Guillem Vives, Vy Pham, Peter Pfeifer, Giuseppe Battaglia and Silvia Muro Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Fri Aug 25 06:20:20 2023 CEST ModDate: Fri Aug 25 06:26:25 2023 CEST Custom Metadata: no Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 16 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 2529767 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- WFVTFE+EURM10 Type 1 Builtin yes yes no 10 0 KBUCWI+URWPalladioL-Bold Type 1 Custom yes yes no 14 0 XDPUXR+URWPalladioL-Roma Type 1 Custom yes yes no 19 0 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HADDNP+PalatinoLinotype TrueType WinAnsi yes yes no 271 0 HADDOB+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 274 0 HADFLH+PalatinoLinotype TrueType MacRoman yes yes no 277 0 FFGMKQ+VnURWPalladioL Type 1 Custom yes yes no 284 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2025-03-12 02:11:13 CET RepresentationInformation: pmc_10531859.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2025-03-12 02:04:09 CET Size: 2529767 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 620 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: Reagents Destination: subsection.2.1 Item: Title: Labeling with 125Iodine Destination: subsection.2.2 Item: Title: NP Preparation Destination: subsection.2.3 Item: Title: NP Coating with Targeting Antibody Destination: subsection.2.4 Item: Title: NP Characterization Destination: subsection.2.5 Item: Title: Encapsulation Efficiency Destination: subsection.2.6 Item: Title: NP Stability and Enzyme Release under Lysosomal Conditions Destination: subsection.2.7 Item: Title: Released Enzyme Activity Destination: subsection.2.8 Item: Title: Statistics Destination: subsection.2.9 Item: Title: Results and Discussion Destination: section.3 Children: Item: Title: Characterization of PLGA NPs Encapsulating HAse Destination: subsection.3.1 Item: Title: Stability under Lysosomal Conditions of PLGA NPs Encapsulating HAse Destination: subsection.3.2 Item: Title: Role of a Targeting Antibody Coat on Stability under Lysosomal Conditions of PLGA NPs Encapsulating HAse Destination: subsection.3.3 Item: Title: Enzyme Release under Lysosomal Conditions from PLGA NPs Encapsulating HAse Destination: subsection.3.4 Item: Title: Catalytic Activity of PLGA NPs Encapsulating HAse Destination: subsection.3.5 Item: Title: Conclusions Destination: section.4 Item: Title: References Destination: section.A. Info: Title: Role of the Lactide:Glycolide Ratio in PLGA Nanoparticle Stability and Release under Lysosomal Conditions for Enzyme Replacement Therapy of Lysosomal Storage Disorders Author: Maria del Moral, Maximilian Loeck, Eameema Muntimadugu, Guillem Vives, Vy Pham, Peter Pfeifer, Giuseppe Battaglia and Silvia Muro Subject: Prior studies demonstrated that encapsulation in poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) enhanced the delivery of enzymes used for replacement therapy (ERT) of lysosomal storage disorders (LSDs). This study examined how the copolymer lactide:glycolide ratio impacts encapsulation, physicochemical characteristics, stability, and release under lysosomal conditions. Hyaluronidase, deficient in mucopolysaccharidosis IX, was encapsulated in NPs synthesized using 50:50, 60:40, or 75:25 lactide:glycolide copolymers. All NPs had diameters compatible with cellular transport (168 nm) and polydispersity indexes (0.16) and -potentials (-35 mV) compatible with colloidal stability. Yet, their encapsulation efficiency varied, with 75:25 NPs and 60:40 NPs having the lowest and highest EE, respectively (15% vs. 28%). Under lysosomal conditions, the 50:50 copolymer degraded fastest (41% in 1 week), as expected, and the presence of a targeting antibody coat did not alter this result. Additionally, 60:40 NPs destabilized fastest (<1 week) because of their smaller diameter, and 75:25 NPs did not destabilize in 4 weeks. All formulations presented burst release under lysosomal conditions (56 78% of the original load within 30 min), with 50:50 and 60:40 NPs releasing an additional small fraction after week 1. This provided 4 weeks of sustained catalytic activity, sufficient to fully degrade a substrate. Altogether, the 60:40 NP formulation is preferred given its higher EE, and 50:50 NPs represent a valid alternative, while the highest stability of 75:25 NPs may impair lysosomes. These results can guide future studies aiming to translate PLGA NP-based ERT for this and other LSDs. 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