Hosseini-Kharat Mahboubeh, Wignall Anthony, Mekonnen Zelalem A, Ung Ben S-Y, Chereda Bradley, Bremmell Kristen E, Grubor-Bauk Branka, Prestidge Clive A
Clinical and Health Sciences, Centre for Pharmaceutical Innovation, University of South Australia, Adelaide, SA 5000, Australia.
Viral Immunology Group, Basil Hetzel Institute for Translational Health Research, Adelaide Medical School, University of Adelaide, Woodville South, SA 5011, Australia.
Pharmaceutics. 2025 Apr 1;17(4):454. doi: 10.3390/pharmaceutics17040454.
: Lipid nanoparticles (LNPs) and polyethyleneimine (PEI) have independently been used for DNA complexation and delivery. However, non-ideal gene delivery efficiency and toxicity have hindered their clinical translation. We developed DNA-PEI-LNPs as a strategy to overcome these limitations and enhance DNA delivery and transgene expression. : Three microfluidic mixing protocols were evaluated: (i) LNPs without PEI, (ii) a single-step process incorporating PEI in the organic phase, and (iii) a two-step process with DNA pre-complexed with PEI before LNP incorporation. The influence of DNA/PEI ratios (1:1, 1:2, 1:3) and DNA/lipid ratios (1:10, 1:40) on particle properties and delivery efficiency was examined. : In luciferase formulations, higher DNA/lipid ratios (1:40) produced smaller particles (136 nm vs. 188 nm) with improved cellular uptake (77% vs. 50%). The two-step method with higher DNA/PEI ratios improved transfection efficiency, with LNP-Luc/PEI 1:3 (40) achieving ~1.9 × 10 relative light units (RLU) in luciferase expression. In green fluorescent protein (GFP) studies, LNP-GFP/PEI 1:3 (40) showed ~23.8% GFP-positive cells, nearly twofold higher than LNP-GFP (40) at ~12.6%. : These results demonstrate the capability of microfluidic-prepared DNA-PEI-LNPs to improve DNA delivery and transgene expression through optimized formulation strategies and selection of appropriate preparation methods.
脂质纳米颗粒（LNPs）和聚乙烯亚胺（PEI）已分别用于DNA复合和递送。然而，不理想的基因递送效率和毒性阻碍了它们的临床转化。我们开发了DNA-PEI-LNPs作为克服这些限制并增强DNA递送和转基因表达的策略。：评估了三种微流控混合方案：（i）不含PEI的LNPs，（ii）在有机相中加入PEI的单步过程，以及（iii）在LNPs掺入之前将DNA与PEI预复合的两步过程。研究了DNA/PEI比率（1:1、1:2、1:3）和DNA/脂质比率（1:10、1:40）对颗粒性质和递送效率的影响。：在荧光素酶制剂中，较高的DNA/脂质比率（1:40）产生了较小的颗粒（136纳米对188纳米），细胞摄取得到改善（77%对50%）。具有较高DNA/PEI比率的两步法提高了转染效率，LNP-Luc/PEI 1:3（40）在荧光素酶表达中达到约1.9×10相对光单位（RLU）。在绿色荧光蛋白（GFP）研究中，LNP-GFP/PEI 1:3（40）显示约23.8%的GFP阳性细胞，比LNP-GFP（40）的约12.6%高出近两倍。：这些结果证明了微流控制备的DNA-PEI-LNPs通过优化配方策略和选择合适的制备方法来改善DNA递送和转基因表达的能力。
