Prasetia I Gnja, Kurniati Neng F, Riani Catur, Mudhakir Diky
Department of Pharmaceutics, School of Pharmacy, Institut Teknologi Bandung (ITB), Bandung, Indonesia.
Pharmacist Profession Study Program, Faculty of Math and Natural Sciences, Universitas Udayana, Bali, Indonesia.
Narra J. 2025 Apr;5(1):e2217. doi: 10.52225/narra.v5i1.2217. Epub 2025 Apr 15.
Familial hypercholesterolemia is a genetic disorder caused by mutations in the low- density lipoprotein receptor gene and the current treatment still focuses on symptom management. The aim of this study was to develop a lipid nanoparticle (LNP)- based delivery system for the CRISPR/Cas9 component in correcting gene mutations. LNPs were prepared using an ultrasonic-solvent emulsification technique by varying the surfactant: oil ratio (SOR), homogenization speed and time, and sonication time. Next, the LNP surface was modified by adding DSPE-PEG-NH and polyethyleneimine. The next stage is to design the single guide RNA (sgRNA) and Donor DNA wildtype (Donor DNA wt). This genetic material was complexed with LNP and then transfected into Hepa1-6 mt cells, an in vitro representation of cells suffering from familial hypercholesterolemia. This optimization process produced LNPs with a particle size of 118.6 ± 0.8 nm and a polydispersity index of 0.34 ± 0.03. The LNP surface modification resulted in a zeta potential of +7.5 mV. A transmission electron microscope (TEM) analysis showed spherical morphology with size distribution following a regular pattern. LNP cell viability tests showed good biocompatibility at concentrations <15 mM with a half-maximal inhibitory concentration (IC) value of 27.7 mM. The dominant cellular uptake mechanism of LNP was through the clathrin-mediated endocytosis (CME) pathway. The Hepa1-6 mt cell model was successfully produced with the transfecting agent Lipofectamine 3000 by homology-directed repair (HDR) mechanism. The LNP-genetic material complex with a ratio of sgRNA:Cas9:Donor DNA wt (1:1:0.04) showed an increase in gene expression of 3.3 ± 0.2 times and protein levels reached 12.95 ± 0.25 ng/mL on day 4 after transfection. The results of this study indicate that the developed LNP-based delivery system has the potential for gene therapy applications in familial hypercholesterolemia.
家族性高胆固醇血症是一种由低密度脂蛋白受体基因突变引起的遗传性疾病，目前的治疗仍侧重于症状管理。本研究的目的是开发一种基于脂质纳米颗粒（LNP）的递送系统，用于递送CRISPR/Cas9组件以纠正基因突变。通过改变表面活性剂与油的比例（SOR）、匀化速度和时间以及超声处理时间，采用超声溶剂乳化技术制备LNP。接下来，通过添加DSPE-PEG-NH和聚乙烯亚胺对LNP表面进行修饰。下一阶段是设计单向导RNA（sgRNA）和供体DNA野生型（供体DNA wt）。将这种遗传物质与LNP复合，然后转染到Hepa1-6 mt细胞中，该细胞是家族性高胆固醇血症细胞的体外模型。该优化过程产生的LNP粒径为118.6±0.8 nm，多分散指数为0.34±0.03。LNP表面修饰导致zeta电位为+7.5 mV。透射电子显微镜（TEM）分析显示为球形形态，尺寸分布呈规则模式。LNP细胞活力测试表明，在浓度<15 mM时具有良好的生物相容性，半数最大抑制浓度（IC）值为27.7 mM。LNP的主要细胞摄取机制是通过网格蛋白介导的内吞作用（CME）途径。通过同源定向修复（HDR）机制，用转染试剂Lipofectamine 3000成功构建了Hepa1-6 mt细胞模型。sgRNA:Cas9:供体DNA wt比例为（1:1:0.04）的LNP-遗传物质复合物在转染后第4天显示基因表达增加3.3±0.2倍，蛋白质水平达到12.95±0.25 ng/mL。本研究结果表明，所开发的基于LNP的递送系统在家族性高胆固醇血症的基因治疗应用中具有潜力。
