Tantan Yasemin, Kaplan Özlem, Bal Kevser, Şentürk Sema, Eker Fidan Emine Büşra, Çelik Sibel, Demir Kamber, Gök Mehmet Koray
Istanbul University-Cerrahpaşa, Faculty of Engineering, Department of Chemical Engineering, Istanbul, Türkiye.
Alanya Alaaddin Keykubat University, Rafet Kayış Faculty of Engineering, Department of Genetics and Bioengineering, Antalya, Türkiye.
Carbohydr Res. 2025 Jan;547:109326. doi: 10.1016/j.carres.2024.109326. Epub 2024 Nov 20.
In this study, we investigated the effect of chitosan modification with tricine on transfection efficiency by preserving its ability to form complexes with plasmid DNA (pDNA) and increasing its hydrophilicity. The inherent limitations of chitosan, such as poor solubility at physiological pH, insufficient cellular uptake, and strong ionic interactions with pDNA, typically result in low transfection efficiency. To overcome these challenges, Tricine, a hydrophilic molecule containing a secondary amine group, was conjugated to chitosan. Chitosan of three different molecular weights (low, medium, and high) was modified with tricine. Structural characterization of the modified chitosan was conducted using Fourier Transformed Infrared Spectroscopy (FTIR) and Nuclear Magnetic Resonance (NMR) analyses. The effects of tricine modification were assessed in terms of hydrophilicity/hydrophobicity, proton buffering capacity, particle size, PDI and zeta potential. Tricine modified low molecular weight chitosan nanoparticles (nLMWChi), which exhibit suitable properties for gene transfer studies, were evaluated regarding pDNA complexation ability, cytotoxicity and in vitro transfection efficiency. The results demonstrated that tricine modification enhanced the gene transfer potential of chitosan, making it competitive with the commercial transfection agent Lipofectamine™ 2000 and offering a promising strategy for non-viral gene therapy applications. Furthermore, the biocompatibility and biodegradability of chitosan, combined with the improved hydrophilicity provided by tricine, makes nLMWChi a safer and more sustainable option for repeated use in gene delivery, overcoming the major limitations associated with other synthetic vectors such as Lipofectamine™ 2000.
在本研究中，我们通过保留壳聚糖与质粒DNA（pDNA）形成复合物的能力并增加其亲水性，研究了用三（羟甲基）甲基甘氨酸修饰壳聚糖对转染效率的影响。壳聚糖存在一些固有局限性，例如在生理pH下溶解度差、细胞摄取不足以及与pDNA的强离子相互作用，这些通常会导致转染效率较低。为了克服这些挑战，将含有仲胺基团的亲水分子三（羟甲基）甲基甘氨酸与壳聚糖共轭。用三（羟甲基）甲基甘氨酸对三种不同分子量（低、中、高）的壳聚糖进行了修饰。使用傅里叶变换红外光谱（FTIR）和核磁共振（NMR）分析对修饰后的壳聚糖进行了结构表征。从亲水性/疏水性、质子缓冲能力、粒径、多分散指数（PDI）和zeta电位方面评估了三（羟甲基）甲基甘氨酸修饰的效果。对具有适合基因转染研究性质的三（羟甲基）甲基甘氨酸修饰的低分子量壳聚糖纳米颗粒（nLMWChi）的pDNA络合能力、细胞毒性和体外转染效率进行了评估。结果表明，三（羟甲基）甲基甘氨酸修饰增强了壳聚糖的基因传递潜力，使其与商业转染试剂Lipofectamine™ 2000具有竞争力，并为非病毒基因治疗应用提供了一种有前景的策略。此外，壳聚糖的生物相容性和生物降解性，结合三（羟甲基）甲基甘氨酸提供的改善的亲水性，使nLMWChi成为在基因递送中重复使用的更安全、更可持续的选择，克服了与其他合成载体（如Lipofectamine™ 2000）相关的主要局限性。
