聚乙烯亚胺（PEI）F25-LMW可使冷冻复合物作为完全活性试剂长期储存，用于RNA干扰介导的基因靶向和DNA递送。

Polyethylenimine PEI F25-LMW allows the long-term storage of frozen complexes as fully active reagents in siRNA-mediated gene targeting and DNA delivery.

作者信息

Höbel Sabrina, Prinz Robert, Malek Anastasia, Urban-Klein Beata, Sitterberg Johannes, Bakowsky Udo, Czubayko Frank, Aigner Achim

机构信息

Department of Pharmacology and Toxicology, Philipps-University School of Medicine, Marburg, Germany.

出版信息

Eur J Pharm Biopharm. 2008 Sep;70(1):29-41. doi: 10.1016/j.ejpb.2008.03.014. Epub 2008 Mar 30.

DOI:10.1016/j.ejpb.2008.03.014

PMID:18499413

Abstract

BACKGROUND

Polyethylenimines (PEIs) are synthetic, charged polymers which function as transfection reagents based on their ability to compact DNA into complexes. Recently, PEI-mediated delivery of nucleic acids has been extended towards small interfering RNAs (siRNAs) which are instrumental in the induction of RNA interference (RNAi). Since RNAi represents a powerful method for specific gene silencing, the PEI-based delivery of siRNAs is a promising tool for novel putative therapeutic strategies.

AIM

For therapeutic use, major requirements are the development of formulations which (i) are sufficiently stable in the presence of serum, and which can be (ii) easily and reproducibly manufactured and (iii) stored for a prolonged time with full retention of their integrity and bioactivity. In this paper, we explore the potential of PEI F25-LMW, a low-molecular weight PEI with superior transfection efficacy and low toxicity, towards these goals.

RESULTS

We have systematically analyzed and determined optimal DNA and siRNA complexation conditions with regard to various parameters including buffer concentration, ionic strength, pH and incubation time. As opposed to 22kDa linear PEI (L-PEI), the low-molecular weight (4-10kDa) PEI F25-LMW performs DNA transfection and siRNA gene targeting with identical efficacies in the presence of serum, thus emphasizing its usefulness in vivo. Furthermore, in contrast to other polyethylenimines, PEI F25-LMW-based DNA or siRNA complexes allow freeze/thawing and frozen storage for several months. Their activity is fully retained without requiring specific buffer conditions or the addition of any lyoprotectant. Physicochemical analysis and atomic force microscopy reveal a distinct size pattern with the presence of two complex subgroups and show that frozen PEI F25-LMW complexes remain stable with little increase in complex size, no changes regarding their zeta potential and cytotoxicity, and full retention of nucleic acid protection.

CONCLUSIONS

Frozen PEI F25-LMW-based complexes represent efficient and stable ready-to-use formulations of DNA- or siRNA-based gene therapy products.

摘要

背景

聚乙烯亚胺（PEIs）是合成的带电荷聚合物，基于其将DNA压缩成复合物的能力而用作转染试剂。最近，PEI介导的核酸递送已扩展到小干扰RNA（siRNAs），其在诱导RNA干扰（RNAi）中起重要作用。由于RNAi是一种强大的特异性基因沉默方法，基于PEI的siRNA递送是新型潜在治疗策略的有前途的工具。

目的

对于治疗用途，主要要求是开发以下制剂：（i）在血清存在下足够稳定，并且（ii）可以容易且可重复地制造，以及（iii）长时间储存并完全保持其完整性和生物活性。在本文中，我们探讨了具有优异转染效率和低毒性的低分子量PEI F25-LMW在实现这些目标方面的潜力。

结果

我们系统地分析并确定了关于各种参数（包括缓冲液浓度、离子强度、pH和孵育时间）的最佳DNA和siRNA复合条件。与22kDa线性PEI（L-PEI）相反，低分子量（4-10kDa）的PEI F25-LMW在血清存在下进行DNA转染和siRNA基因靶向的效率相同，从而强调了其在体内的实用性。此外，与其他聚乙烯亚胺相比，基于PEI F25-LMW的DNA或siRNA复合物允许冻融和冷冻储存数月。它们的活性完全保留，无需特定的缓冲条件或添加任何冻干保护剂。物理化学分析和原子力显微镜揭示了存在两个复合亚组的独特尺寸模式，并表明冷冻的PEI F25-LMW复合物保持稳定，复合物尺寸几乎没有增加，其zeta电位和细胞毒性没有变化，并且完全保留了核酸保护作用。