Introduction: Glial cell line-derived neurotrophic factor (GDNF) is a well-establish therapeutic agent for Parkinson’s disease (PD) 1. Its therapeutic potential, being a glycosylated protein, will largely depend on the possibility of administering it with a glycosylation pattern similar to the one present in the native protein 2. Moreover, although several approaches to deliver this protein to the brain have been described 3, a promising strategy would be the use of nanoparticles (NPs) containing GDNF in the dopamine-depleted brain areas. Therefore, the aim of this work was to develop and characterize biodegradable NPs loaded with recombinant GDNF produced in mammalian cells for brain tissue engineering.
Methods: GDNF was produced by our group 4. GDNF purity was visualized by SDS-PAGE followed by Coomasie Blue (CB) staining. PC12 cell-based bioassay was used to assess its bioactivity. GDNF was encapsulated in polymeric NPs by multiple emulsion solvent evaporation method using Total Recirculation One Machine System (TROMS). Dynamic light scattering was used to measure the mean particle diameter and polydispersity index (PDI). Encapsulation efficiency was quantified by ELISA. NPs morphology was characterized by scanning electron microscopy (SEM). GDNF in vitro release from NPs disrupted with DMSO was determined by ELISA.
Results: GDNF was highly pure. The NPs showed a diameter of 405.5 ± 2.9 nm and a PDI of 0.08 ± 0.03. The encapsulation efficiency was 61.6%. SEM analysis showed spherical particles. GDNF released within the first 24 hours was 19.10 ± 3.5%, followed by a phase of sustained-release with 50.6 ± 3.1% of hGDNF being released within 28 days. PC12 treatment with the hGDNF released from NPs induced outgrowth of neurites in these cells indicating that hGDNF remains bioactive after its nanoencapsulation.
Conclusions: GDNF-NPs has been successfully prepared using TROMS, showing a high protein loading and a sustained release. The developed nanosystem has great potential for brain tissue engineering applications.
Acknowledgements
- Torres thanks the Spanish Ministry of Education (Programa FPU (FPU17/01212)) and Government of Navarra (2019_66_NAB9). E. Garbayo is supported by a “Ramon y Cajal Fellowship (RYC2018-025897-I).
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