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Formulation of a Peptide Nucleic Acid Based Nucleic Acid Delivery Construct

机译:基于肽核酸的核酸递送构建体的配制

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Gene delivery biomaterials need to be designed to efficiently achieve nuclear delivery of plasmid DNA. Polycations have been used to package DNA and other nucleic acids within submicrometer-sized particles, offering protection from shear-induced or enzymatic degradation. However, cytotoxicity issues coupled with limited in vivo transfection efficiencies minimize the effectiveness of this approach. In an effort to improve upon existing technologies aimed at delivering nucleic acids, an alternative approach to DNA packaging was explored. Peptide nucleic acids (PNAs) were used to directly functionalize DNA with poly(ethylene glycol) (PEG) chains that provide a steric layer and inhibit multimolecular aggregation during complexation. DNA prePEGylation by this strategy was predicted to enable the formation of more homogeneous and efficiently packaged polyplexes. In this work, DNA-PNA-peptide-PEG (DP3) conjugates were synthesized and self-assembled with 25 kDa poly(ethylenimine) (PEI). Complexes with small standard deviations and average diameters ranging 30−50 nm were created, with minimal dependence of complex size on N/P ratio (PEI amines to DNA phosphates). Furthermore, PEI−DNA interactions were altered by the derivatization strategy, resulting in tighter compaction of the PEI−DP3 complexes in comparison to PEI−DNA complexes. Transfection experiments in Chinese hamster ovary (CHO) cells revealed comparable transfection efficiencies but reduced cytotoxicities of the PEI−DP3 complexes relative to PEI−DNA complexes. The enhanced cellular activities of the PEI−DP3 complexes were maintained following the removal of free PEI from the PEI−DP3 formulations, whereas the cellular activity of the conventional PEI−DNA formulations was reduced by free PEI removal. These findings suggest that DNA prePEGylation by the PNA-based strategy might provide a way to circumvent cytotoxicity and formulation issues related to the use of PEI for in vivo gene delivery.
机译:基因传递生物材料需要进行设计,以有效地实现质粒DNA的核传递。聚阳离子已被用于将DNA和其他核酸包装在亚微米大小的颗粒中,从而提供保护以防止剪切诱导的降解或酶促降解。但是,细胞毒性问题以及有限的体内转染效率使这种方法的有效性最小化。为了改进旨在递送核酸的现有技术,探索了DNA包装的替代方法。肽核酸(PNA)用于直接用聚乙二醇(PEG)链对DNA进行功能化,该聚乙二醇提供空间层并在复合过程中抑制多分子聚集。预测通过该策略进行的DNA预聚乙二醇化可形成更均一且有效包装的多链体。在这项工作中,合成了DNA-PNA-肽-PEG(DP3)共轭物,并用25 kDa的聚乙烯亚胺(PEI)自组装。创建的复合物的标准偏差小,平均直径范围为30-50 nm,而复合物大小对N / P比(PEI胺与DNA磷酸酯)的依赖性最小。此外,派生策略改变了PEI-DNA的相互作用,与PEI-DNA配合物相比,导致PEI-DP3配合物更加紧密。在中国仓鼠卵巢(CHO)细胞中进行转染实验显示,与PEI-DNA复合物相比,PEI-DP3复合物的转染效率相当,但细胞毒性降低。从PEI-DP3制剂中除去游离PEI后,PEI-DP3复合物的细胞活性得以维持,而常规PEI-DNA制剂的细胞活性却因游离PEI的除去而降低。这些发现表明,通过基于PNA的策略进行的DNA预聚乙二醇化可能提供一种途径来规避与使用PEI进行体内基因传递有关的细胞毒性和制剂问题。

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