In vitro treatment of congenital disorder of glycosylation type Ia using PLGA nanoparticles loaded with GDP‑Man

Bortot,Barbara; De Martino,Eleonora; Tesser,Alessandra; Ura,Blendi; Ruozi,Barbara; Aloisio,Michelangelo; Biffi,Stefania; Addobbati,Riccardo; Tosi,Giovanni; Dolcetta,Diego; Severini,Giovanni   Maria

doi:10.3892/ijmm.2019.4199

In vitro treatment of congenital disorder of glycosylation type Ia using PLGA nanoparticles loaded with GDP‑Man

Authors:
- Barbara Bortot
- Eleonora De Martino
- Alessandra Tesser
- Blendi Ura
- Barbara Ruozi
- Michelangelo Aloisio
- Stefania Biffi
- Riccardo Addobbati
- Giovanni Tosi
- Diego Dolcetta
- Giovanni Maria Severini
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Affiliations: Institute for Maternal and Child Health‑IRCCS ‘Burlo Garofolo’, I‑34137 Trieste, Italy, DSM, Department of Medical Sciences, University of Trieste, I‑34149 Trieste, Italy, Department of Life Sciences, University of Modena and Reggio Emilia, I‑41121 Modena, Italy, UOSD SAFU, RiDAIT Department, The Regina Elena National Cancer Institute, I‑00144 Rome, Italy
Published online on: May 16, 2019 https://doi.org/10.3892/ijmm.2019.4199
Pages: 262-272

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Abstract

Congenital disorder of glycosylation (CDG) type Ia is a multisystem disorder that occurs due to mutations in the phosphomannomutase 2 (PMM2) gene, which encodes for an enzyme involved in the N‑glycosylation pathway. Mutated PMM2 leads to the reduced conversion of mannose‑6‑P to mannose‑1‑P, which results in low concentration levels of guanosine 5'‑diphospho‑D‑mannose (GDP‑Man), a nucleotide‑activated sugar essential for the construction of protein oligosaccharide chains. In the present study, an in vitro therapeutic approach was used, based on GDP‑Man‑loaded poly (D,L‑lactide‑co‑glycolide) (PLGA) nanoparticles (NPs), which were used to treat CDG‑Ia fibroblast cultures, thus bypassing the glycosylation pathway reaction catalysed by PMM2. To assess the degree of hypoglycosylation in vitro, the present study examined the activities of α‑mannosidase, β‑glucoronidase and β‑galactosidase in defective and normal fibroblasts. GDP‑Man (30 µg/ml GDP‑Man PLGA NPs) was incubated for 48 h with the cells and the specific activities of α‑mannosidase and β‑galactosidase were estimated at 69 and 92% compared with healthy controls. The residual activity of β‑glucoronidase increased from 6.5 to 32.5% and was significantly higher compared with that noted in the untreated CDG‑Ia fibroblasts. The glycosylation process of fibroblasts was also analysed by two‑dimensional electrophoresis. The results demonstrated that treatment caused the reappearance of several glycosylated proteins. The data in vitro showed that GDP‑Man PLGA NPs have desirable efficacy and warrant further evaluation in a preclinical validation animal model.

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