Raimbekova Aigul, Kart Ulpan, Yerishova Akbayan, Elebessov Timur, Yegorov Sergey, Pham Tri Thanh, Hortelano Gonzalo
Department of Biology, School of Sciences and Humanities, Nazarbayev University, Astana, Kazakhstan.
PeerJ. 2025 Jan 24;13:e18750. doi: 10.7717/peerj.18750. eCollection 2025.
Chitosan nanoparticles (CsNPs) are an effective and inexpensive approach for DNA delivery into live cells. However, most CsNP synthesis protocols are not optimized to allow long-term storage of CsNPs without loss of function. Here, we describe a protocol for CsNP synthesis, lyophilization, and sonication, to store CsNPs and maintain transfection efficiency.
The size and zeta potential of CsNPs were analyzed by dynamic light scattering (DLS) and the morphology of CsNPs was assessed by transmission electron microscopy (TEM). HEK293 cells were transfected with CsNPs, and expression of H2B-CMV-mScarlet plasmid was assessed by flow cytometry. Confocal microscopy was used to visualize post-transfection gene expression. Time, volume, and effect of sonication were tested to optimize the lyophilization process.
DLS and TEM analysis indicated amine groups on chitosan to phosphate groups on DNA (N:P) ratios yielded smaller CsNPs sizes. Transfection efficiency, measured by FACS and confocal microscopy, peaked at N:P ratios of 2:1 and 3:1 for both fresh and lyophilized CsNPs. Chitosan/DNA complexes remained stable in solution for at least 72 h at a ratio ≥2:1 as assessed by agarose gel electrophoresis. A lower surface charge with lower N:P ratios was indicated by zeta potential measurements. Lyophilized CsNPs lost 50% transfection efficiency compared to those freshly made. In contrast, sonication of lyophilized CsNPs restored their transfection efficiency to the level of fresh CsNPs. Sonicated CsNPs maintained spherical morphology, while unsonicated CsNPs showed aggregates. Cytotoxicity assays revealed high cell viability (>90%) after CsNPs transfection for a ratio of 2:1 or 3:1.
This optimized CsNPs synthesis protocol opens the possibility of long-term storage for CsNPs, which would provide broader applications of this technology.
壳聚糖纳米颗粒（CsNPs）是一种将DNA导入活细胞的有效且廉价的方法。然而，大多数CsNP合成方案并未经过优化，无法实现CsNPs的长期储存而不丧失功能。在此，我们描述了一种CsNP合成、冻干和超声处理的方案，以储存CsNPs并维持转染效率。
通过动态光散射（DLS）分析CsNPs的大小和zeta电位，并通过透射电子显微镜（TEM）评估CsNPs的形态。用CsNPs转染HEK293细胞，并通过流式细胞术评估H2B-CMV-mScarlet质粒的表达。共聚焦显微镜用于观察转染后基因的表达。测试超声处理的时间、体积和效果以优化冻干过程。
DLS和TEM分析表明，壳聚糖上的胺基与DNA上的磷酸基团之比（N:P）产生较小的CsNP尺寸。通过FACS和共聚焦显微镜测量，新鲜和冻干的CsNPs在N:P比为2:1和3:1时转染效率达到峰值。通过琼脂糖凝胶电泳评估，壳聚糖/DNA复合物在溶液中以≥2:1的比例至少稳定72小时。zeta电位测量表明，较低的N:P比具有较低的表面电荷。与新鲜制备的CsNPs相比，冻干的CsNPs失去了50%的转染效率。相比之下，冻干CsNPs的超声处理将其转染效率恢复到新鲜CsNPs的水平。超声处理的CsNPs保持球形形态，而未超声处理的CsNPs则显示聚集。细胞毒性试验显示，在CsNPs以2:1或3:1的比例转染后，细胞活力较高（>90%）。
这种优化的CsNP合成方案为CsNPs的长期储存提供了可能性，这将使该技术得到更广泛的应用。
