Physicochemical Characterization and Microfluidics-Assisted Synthesis of Ligand- Functionalized Lipid–Polymer Hybrid Nanoparticles for Targeted Delivery of siRNA Across the Blood–Brain Barrier in Glioblastoma Therapy

Abstract

Background:
Glioblastoma (GBM) is an aggressive and highly invasive brain tumor with poor prognosis, largely
due to the presence of the blood–brain barrier (BBB), which limits the effective delivery of
therapeutic agents. Small interfering RNA (siRNA)-based therapies offer a promising strategy for
targeted gene silencing; however, their clinical application is hindered by instability, poor cellular
uptake, and limited BBB permeability.
Objective:
This study aimed to develop and characterize ligand-functionalized lipid–polymer hybrid
nanoparticles (LPHNs) using microfluidics-assisted synthesis for efficient and targeted delivery of
siRNA across the BBB in glioblastoma therapy.
Methods:
LPHNs were synthesized using a microfluidic platform by optimizing flow rate ratio (FRR) and total
flow rate (TFR). Nanoparticles were functionalized with targeting ligands (e.g., transferrin) and
loaded with siRNA targeting oncogenes (EGFR/VEGF). Physicochemical properties including
particle size, polydispersity index (PDI), zeta potential, morphology, and stability were evaluated. In
vitro studies included cytotoxicity assays, cellular uptake analysis, BBB permeability assessment
using a Transwell model, and gene silencing evaluation via qRT-PCR and Western blot. In vivo
studies were conducted in an orthotopic glioblastoma model to assess biodistribution, therapeutic
efficacy, survival, and toxicity.
Results:
Optimized LPHNs exhibited a particle size of ~100 nm with low PDI (<0.2) and stable zeta potential
(~−18 mV). High siRNA encapsulation efficiency (~87%) and excellent stability were observed.
Ligand-functionalized nanoparticles demonstrated significantly enhanced cellular uptake (2.5-fold
increase) and BBB permeability compared to non-targeted systems. Gene silencing efficiency reached
~70–80% reduction in target gene expression. In vivo studies revealed preferential brain tumor
accumulation, significant tumor regression, and improved median survival (52 days vs. 28 days in
controls) without notable systemic toxicity.
Conclusion:
Microfluidics-assisted synthesis of ligand-functionalized LPHNs provides a robust and efficient
platform for targeted siRNA delivery across the BBB. The developed system significantly enhances
therapeutic efficacy in glioblastoma, demonstrating strong potential for clinical translation in neuro-
oncology.