Delivery of messenger RNA (mRNA) using lipid nanoparticles (LNPs) is expected to be applied to various diseases following the successful clinical use of the mRNA COVID-19 vaccines. This study aimed to evaluate the effect of the cholesterol molar percentage of mRNA-LNPs on protein expression in hepatocellular carcinoma-derived cells and in the liver after intramuscular or subcutaneous administration of mRNA-LNPs in mice. For mRNA-LNPs with cholesterol molar percentages reduced to 10 mol% and 20 mol%, we formulated neutral charge particles with a diameter of approximately 100 nm and polydispersity index (PDI) <0.25. After the intramuscular or subcutaneous administration of mRNA-LNPs with different cholesterol molar percentages in mice, protein expression in the liver decreased as the cholesterol molar percentage in mRNA-LNPs decreased from 40 mol% to 20 mol% and 10 mol%, suggesting that reducing the cholesterol molar percentage in mRNA-LNPs decreases protein expression in the liver. Furthermore, in HepG2 cells, protein expression decreased as cholesterol in mRNA-LNPs was reduced by 40 mol%, 20 mol%, and 10 mol%. These results suggest that the downregulated expression of mRNA-LNPs with low cholesterol content in the liver involves degradation in systemic circulating blood and decreased protein expression after hepatocyte distribution.

To achieve the desired delivery effect, extracellular vesicles (EVs) must bypass rapid clearance from circulation and exhibit affinity for target cells; however, it is difficult to simultaneously incorporate two materials into EVs. Post-insertion is a general modification method that can be performed by simply mixing different solutions. Previously, we have developed a microfluidic post-insertion method that supported fast and upscaled modification of EVs using KK-modified high-functionality and -quality (HFQ) lipids. Here, we used microfluidic post-insertion to achieve simultaneous incorporation of polyethylene glycol (PEG) lipids and KK or RGD-modified HFQ lipids into milk-derived EVs to avoid uptake from the reticuloendothelial system and increase the uptake into target cells. PEG lipid and HFQ lipids were formulated to produce micelles and subsequently mixed with EV solution using a microfluidic device. Compared to bulk mixing, microfluidic post-insertion showed higher cellular association. Altered cellular association capacities and endocytic pathways indicated simultaneous incorporation. The cellular association of modified EVs can be adjusted by altering the ratio of (EK)₄-KK in micelles with slight changes in physicochemical properties. Furthermore, microfluidic post-insertion is also suitable for (SG)₅-RGD, which is insoluble in phosphate-buffered saline (PBS). Our results may be valuable for the development and manufacture of functional EVs as drug delivery systems for clinical applications.

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.