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.

Lipid nanoparticles (LNPs) have emerged as a key delivery platform for mRNA therapeutics, as demonstrated by the clinical success of mRNA vaccines against SARS-CoV-2. LNPs are now being investigated for various applications, such as cancer immunotherapy and the treatment of genetic disorders. However, LNPs tend to accumulate predominantly in the liver and lack intrinsic cell-type specificity. Modifying LNPs with antibodies is a promising strategy for enhancing cell-specific delivery. We have previously developed orientation-controlled anti-transferrin receptor and anti-PD-L1 antibody modifications for the targeted delivery of nucleic acids, including mRNA and siRNA. In this study, we newly applied this platform to trastuzumab, a clinically established high-affinity anti-HER2 therapeutic antibody, to develop orientation-controlled trastuzumab-modified mRNA/LNPs for HER2-selective delivery. we evaluated trastuzumab-modified mRNA/LNPs in HER2-overexpressing SKOV-3 ovarian cancer cells and HER2-low MDA-MB-231 breast cancer cells. Trastuzumab-modified mRNA/LNPs exhibited over 100-fold greater cellular association than unmodified LNPs in cultured HER2-overexpressing SKOV-3 cells. Furthermore, after intratumoral administration in a mouse model bearing subcutaneous SKOV-3 tumors, the trastuzumab-modified group showed significantly higher luciferase expression than the unmodified and isotype-controlled antibody-modified groups. These findings support FcBP-HFQ lipid-mediated trastuzumab modification of LNPs as a useful approach for efficient cell-selective mRNA delivery.

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.