Resveratrol-Loaded PLGA Enhanced Vitrified Oocyte Viability through Rab11fip4/Arf5-Mediated Internalization Route

Irreversible injury accompanied by oocyte vitrification
is still
challenging. Recent advances in nanotechnology offer promising therapeutic
potential. However, the molecular mechanisms by which nanomaterials
protect vitrified oocytes remain elusive. In this study, resveratrol-loaded
poly(lactic-co-glycolic acid) nanoparticles (PLGA-RES)
were synthesized, and the successful synthesis was confirmed by the
results of Fourier transform infrared spectroscopy (FTIR) characterization.
Thereafter, the effect of PLGA-RES on the quality of vitrified oocytes
was explored. Our results showed that the synthesized PLGA-RES nanoparticles,
with an average size of 163.5 nm, demonstrated time- and dose-dependent
internalization in mouse oocytes. Transmission electron microscopy
(TEM) analysis revealed their distribution around the membrane and
perinuclear region. Treatment with PLGA-RES significantly increased
the germinal vesicle breakdown (GVBD) rate and reduced the aneuploidy
rate in vitrified oocytes. Transcriptome analysis identified 447 upregulated
and 185 downregulated mRNAs, with the endocytosis pathway playing
a crucial role in PLGA-RES regulation. Short time-series expression
miner analysis (STEM) and weighted gene coexpression network analysis
(WGCNA) further identified Rab11fip4 as a core gene in the PLGA-RES
internalization, which was also confirmed by a molecular docking study.
Rab11fip4 was further found to interact with Arf5 via coimmunoprecipitation
(co-IP) analysis. Knockdown of Rab11fip4 significantly impaired oocyte
maturation and nanoparticles internalization. Further experiments
showed that the internalization of PLGA-RES was decreased by endocytic
inhibitors, and the Rab11fip4-mediated recycling endosome pathway
could protect PLGA-RES from lysosomal degradation. In summary, PLGA-RES
could improve vitrified oocyte viability through the endocytic pathway,
highlighting the importance of Rab11fip4 in nanoparticle internalization
and functional modulation.