Background/Objectives: Glioblastoma (GBM) is an aggressive primary brain tumor with limited therapeutic options despite multimodal treatment. Small interfering RNA (siRNA)-based therapeutics can silence tumor-promoting genes, but achieving efficient and tumor-specific delivery remains challenging. Lipid nanoparticles (LNPs) are promising siRNA carriers; however, conventional antibody conjugation can impair antigen recognition and complicate manufacturing. This study aimed to establish a modular Fc-binding peptide (FcBP)-mediated post-insertion strategy to enable PD-L1-targeted delivery of VEGF-siRNA via LNPs for GBM therapy. Methods: Preformed VEGF-siRNA-loaded LNPs were functionalized with FcBP–lipid conjugates, enabling non-covalent anchoring of anti-PD-L1 antibodies through Fc interactions. Particle characteristics were analyzed using dynamic light scattering and encapsulation efficiency assays. Targeted cellular uptake and VEGF gene silencing were evaluated in PD-L1-positive GL261 glioma cells. Anti-tumor efficacy was assessed in a subcutaneous GL261 tumor model following repeated intratumoral administration using tumor volume and bioluminescence imaging as endpoints. Results: FcBP post-insertion preserved LNP particle size (125.2 ± 1.3 nm), polydispersity, zeta potential, and siRNA encapsulation efficiency. Anti-PD-L1–FcBP-LNPs significantly enhanced cellular uptake (by ~50-fold) and VEGF silencing in PD-L1-expressing GL261 cells compared to controls. In vivo, targeted LNPs reduced tumor volume by 65% and markedly suppressed bioluminescence signals without inducing weight loss. Final tumor weight was reduced by 63% in the anti-PD-L1–FcBP–LNP group (656.9 ± 125.4 mg) compared to the VEGF-siRNA LNP group (1794.1 ± 103.7 mg). The FcBP-modified LNPs maintained antibody orientation and binding activity, enabling rapid functionalization with targeting antibodies. Conclusions: The FcBP-mediated post-insertion strategy enables site-specific, modular antibody functionalization of LNPs without compromising physicochemical integrity or antibody recognition. PD-L1-targeted VEGF-siRNA delivery demonstrated potent, selective anti-tumor effects in GBM murine models. This platform offers a versatile approach for targeted nucleic acid therapeutics and holds translational potential for treating GBM.

Various functionalization methods have been explored to enhance the targeted delivery of small extracellular vesicles (sEVs). We recently developed a microfluidic post-insertion method that enables the simultaneous incorporation of polyethylene glycol (PEG) lipids and high-functionality and high-quality (HFQ) lipids into bovine milk-derived sEVs. Since sEVs from different sources differ in surface composition and properties that significantly impact modifications, it is crucial to determine whether this method is applicable to sEVs from other origins. In this study, we used a PEG-lipid and a peptide consisting of arginine-glycine-aspartic acid (RGD) linked to a serine-glycine repeat (SG)₅ to modify sEVs derived from mouse macrophage cells (RAW264.7) and compared the results with those obtained using the conventional bulk mixing method in vitro. The RGD-PEG sEVs prepared via microfluidic post-insertion and bulk mixing exhibited similar physicochemical properties and cellular binding functions. Successful incorporation of both lipids was confirmed by changes in physicochemical properties, cellular binding capacity, and endocytosis pathways. The results indicated that PEG reduced overall uptake, while RGD facilitated cellular association of RGD-PEG sEVs compared to PEG sEVs alone. Our approach for the simultaneous incorporation of PEG and HFQ lipids may open new avenues for the functional application of sEVs derived from diverse origins.