穿膜肽增强聚乙烯亚胺介导的基因转染效率的机制研究

目的研究穿膜肽增强支链聚乙烯亚胺（BPEI）在CHO-K1细胞中的基因转染效率的机制。方法分别合成蜂毒肽（melittin）及其疏水核心区MT20,利用圆二色光谱（CD）分析其二级结构;通过溶血试验比较二者穿透细胞膜的能力;加入蜂毒肽和MT20前后,观察钙黄绿素（calcein）在HeLa细胞内的分布情况。分别将蜂毒肽或MT20与BPEI按一定比例混合,以荧光素酶（luciferase）为报告基因,检测荧光素酶在CHO-K1细胞中的转染效率;以MTT法检测基因转染后的细胞毒性。结果在模拟膜环境的甲醇溶液中,蜂毒肽和MT20都呈现一定的α-螺旋结构,比例分别为59.63%和35.67%,说明其二级结构与之穿膜功能相关;蜂毒肽只能在中性条件下穿透细胞膜导致红细胞溶血,而MT20在中性和酸性条件下都具有穿膜能力;加入蜂毒肽和MT20后能够促进钙黄绿素在MT20组中呈弥散分布,在蜂毒肽组中呈颗粒状分布并且在核仁内蓄积;MT20和蜂毒肽都能够增强BPEI在CHO-K1细胞中的基因转染效率,其中MT20的增强作用更强且细胞毒性较蜂毒肽以及单独使用BPEI和Lipofectamine 2000时要低。结论蜂毒肽的疏水核心区MT20通过增加PEI/DNA复合物颗粒从内含体逃逸并促进其进入细胞核而增加其基因转染效率,是一种低毒的基因转染增强剂,有可能在难以转染的细胞系中发挥重要作用。     

Exploiting protein cationization techniques in future drug development

The development of a method for the efficient intracellular delivery of inherently non-permeable proteins is needed for manipulation of cellular phenotypes or the discovery of protein-based drugs. It has been demonstrated that proteins artificially cationized by chemical conjugation show efficient intracellular delivery via adsorptive-mediated endocytosis and then can exert their biological activity in cells. Studies have also revealed that cationic peptides known as cell-penetrating peptides (CPPs) provide a means to deliver molecules into mammalian cells. Although the internalization mechanisms remain controversial, it is now becoming clear that the main port of entry into cells by CPPs also involves adsorptive-mediated endocytosis rather than the direct penetration of the plasma membrane. As the mammalian cell membrane possesses an abundance of negatively charged glycoproteins and glycosphingolipids, cationization of proteins is a reasonable choice to endow them with the ability for intracellular delivery. Cationization of proteins is usually accompanied by drastic changes in protein properties, structure and biological activities. Recently developed sophisticated protein chemistry can minimize these side effects. Therefore, protein cationization techniques will hopefully prove to be powerful tools for innovative research and drug discovery. In this review, techniques for cationization of proteins and their intracellular delivery, as well as some of their potential therapeutic applications, are discussed.     

Myristic acid-conjugated polyethylenimine for brain-targeting delivery: in vivo and ex vivo imaging evaluation

To investigate the potential of myristic acid (MC) to mediate brain delivery of polyethylenimine (PEI) as a gene delivery system, a covalent conjugate (MC-PEI) of MC, and PEI was synthesized. A near-infrared fluorescence probe, IR820 was conjugated to MC-PEI to explore its in vivo distribution after intravenous (i.v.) administration in mice. The brain targeting ability of MC-PEI was evaluated by near-infrared fluorescence imaging and analyzed semiquantitatively by fluorescence intensity, respectively. Significant NIR fluorescent signal was detected in the brain 12?h after i.v. administration and further confirmed by imaging the whole brain and brain slices. Semiquantitative results from fluorescence intensity further supported the successful brain delivery of MC-PEI which led to a very significant increase (～200%) in the brain uptake after i.v. injection in comparison with unmodified PEI. The capability of MC-PEI to condense DNA was further confirmed using agarose gel retardation assay, indicating its potential for gene delivery. The significant in vivo and ex vivo results suggest that MC-PEI is a promising brain-targeting drug delivery system, especially for gene delivery.     

Comparison of the efficiency and toxicity of sonoporation with branched polyethylenimine-mediated gene transfection in various cultured cell lines

Objective. The objective of this study is to evaluate transfection efficiency and safety for gene delivery by sonoporation in comparison with cationic polymer gene carrier branched polyethylenimine (BPEI).

Methods. The cDNA expressing VEGF¹⁶⁵ was cloned under chicken β-actin promoter. The plasmid DNA was transfected into the CHO, HEK293, and NIH3T3 cells using microbubble-based sonoporation and BPEI (25 kDa) under various conditions. Enzyme-linked immunosorbent assay (ELISA) was used to determine the expressed protein level. Cytotoxicities of transfection methods were compared by Cell Counting Kit-8.

Results. At 1 MHz intensity, transfection efficiency of sonoporation was enhanced by microbubble concentration with no detrimental effects. By contrast, BPEI exacerbated cell viability, despite its high transgene expression efficiency.

Conclusion. Sonoporation gene therapy might be the safest technique to be used in actual clinical practice.     

DNA delivery to the mitochondria sites using mitochondrial leader peptide conjugated polyethylenimine

Some genetic diseases are associated with the defects of the mitochondrial genome. Direct DNA delivery to the mitochondrial matrix has been suggested as an approach for mitochondrial gene therapy for these diseases. We hypothesized that a mitochondrial leader peptide (LP) conjugated polyethylenimine (PEI) could deliver DNA to the mitochondrial sites. PEI-LP was synthesized by the conjugation of LP to PEI using disulfide bond. The complex formation of PEI-LP with DNA was confirmed by a gel retardation assay. In this study, DNA was completely retarded at a 0.4/1 PEI-LP/DNA weight ratio. In vitro delivery tests into isolated mitochondria or living cells were performed with rhodamin-labeled DNA and PEI-LP. In vitro cell-free delivery assay with isolated mitochondria showed that PEI-LP/DNA complexes were localized at mitochondria sites. Furthermore, the PEL-LP/DNA complexes were localized at the mitochondrial sites in living cells. However, a control carrier, PEI, did not show this effect. In addition, MTT assay showed that PEI-LP showed lower cytotoxicity than PEI. These results suggest that PEI-LP can deliver DNA to the mitochondrial sites and may be useful for the development of mitochondrial gene therapy.     

The efficiency of a novel delivery system (PEI600-Tat) in development of potent DNA vaccine using HPV16 E7 as a model antigen

DNA vaccination is a promising approach for inducing both humoral and cellular immune responses. The mode of plasmid DNA delivery is critical to make progress in DNA vaccination. Using human papillomavirus type 16 E7 as a model antigen, this study evaluated the effect of peptide-polymer hybrid including PEI600-Tat conjugate as a novel gene delivery system on the potency of antigen-specific immunity in mice model. At ratio of 10:50 PEI-Tat/E7DNA (w/w), both humoral and cellular immune responses were significantly enhanced as compared with E7DNA construct and induced Th1 response. Therefore, this new delivery system could have promising applications in gene therapy.     

Biophysical and Structural Characterization of Polyethylenimine-Mediated siRNA Delivery in Vitro

Purpose The goals of this study were as follows: 1) to evaluate the efficacy of different polyethylenimine (PEI) structures for siRNA delivery in a model system, and 2) to determine the biophysical and structural characteristics of PEI that relate to siRNA delivery.Materials and Methods Biophysical characterization (effective diameter and zeta potential), cytotoxicities, relative binding affinities and in vitro transfection efficiencies were determined using nanocomplexes formed from PEI's of 800, 25,000, (both branched) and 22,000 (linear) molecular weights at varying N:P ratios and siRNA concentrations. The HR5-CL11 cell line stably expressing luciferase was used as a model system in vitro.Results Successful siRNA delivery was observed within a very narrow window of conditions, and only with the 25,000 branched PEI at an N:P ratio of 6:1 and 8:1 and with 200 nM siRNA. While the zeta potential and size of PEI:siRNA complexes correlated to transfection efficacy in some cases, complex stability may also affect transfection efficacy.Conclusions The ability of PEI to transfer functionally active siRNA to cells in culture is surprisingly dependent on its biophysical and structural characteristics when compared to its relative success and ease of use for DNA delivery.     

Full deacylation of polyethylenimine dramatically boosts its gene delivery efficiency and specificity to mouse lung

High-molecular-mass polyethylenimines (PEIs) are widely used vectors for nucleic acid delivery. We found that removal of the residual N-acyl moieties from commercial linear 25-kDa PEI enhanced its plasmid DNA delivery efficiency 21 times in vitro, as well as 10,000 times in mice with a concomitant 1,500-fold enhancement in lung specificity. Several additional linear PEIs were synthesized by acid-catalyzed hydrolysis of poly(2-ethyl-2-oxazoline), yielding the pure polycations. PEI87 and PEI217 exhibited the highest efficiency in vitro: 115-fold and 6-fold above those of the commercial and deacylated PEI25s, respectively; moreover, PEI87 delivered DNA to mouse lung as efficiently as the pure PEI25 but at a lower concentration and with a 200-fold lung specificity. These improvements stem from an increase in the number of protonatable nitrogens, which presumably results in a tighter condensation of plasmid DNA and a better endosomal escape of the PEI/DNA complexes. As a validation of the potential of such linear, fully deacylated PEIs in gene therapy for lung diseases, systemic delivery in mice of the complexes of a short interfering RNA (siRNA) against a model gene, firefly luciferase, and PEI25 or PEI87 afforded a 77% and 93% suppression of the gene expression in the lungs, respectively. Furthermore, a polyplex of a siRNA against the influenza viral nucleocapsid protein gene and PEI87 resulted in a 94% drop of virus titers in the lungs of influenza-infected animals.     

Efficient Gene Delivery to Myocardium with Ultrasound Targeted Microbubble Destruction and Polyethylenimine

The aim of present study was to evaluate the feasibility and efficiency of enhanced green fluorescent protein （EGFP） gene delivery to myocardium in vivo by ultrasound targeted microbubble destruction （UTMD） and polyethylenimine （PEI）. SonoVue/DNA and PEI/DNA/SonoVue complexes were prepared. Gel electrophoresis analysis was performed to determine the structural integrity of plasmid DNA or PEI/DNA after UTMD. Solutions of plasmid DNA, SonoVue/DNA, PEI/DNA complexes or PEI/DNA/SonoVue complexes were respectively transduced into BALB/c mice hearts by means of transthoracic ultrasound irradiation. Mice undergoing PBS injection, plasmid injection or PEI/DNA complexes injection without ultrasound irradiation served as controls. Gene expression in myocardium was detected 4 days after treatment. Cryosections and histological examinations were conducted. Electrophoresis gel assay showed no damage to DNA or PEI/DNA complexes after UTMD. When the heart was not exposed to ultrasound, the expression of EGFP was observed in the subendocardial myocardium obviously. The strongest expression was detected in the anterior wall of the left ventricle when the heart was exposed to ultrasound alone. Injection of PEI/DNA complexes and UTMD resulted in the highest transfection efficiency and the distributional difference of EGFP was not obvious. No tissue damage was seen histologically. In conclusion, a combination of UTMD and PEI was highly effective in transfecting mice hearts without causing any apparently adverse effect. It provides an alternative to current clinical gene therapy and opens a new concept of non-viral gene delivery for the treatment of cardiac disease.     

非病毒型载体介导基因转染

基因载体是制约基因转移技术发展的关键。近年来,非病毒载体由于其安全、低毒、低免疫原性等特点而备受青睐。文章以脂质体和聚乙烯亚胺为代表,介绍了非病毒载体的性质、介导转染的机制。随着人们对细胞转染机制了解的深入以及生物材料科学的迅速发展,非病毒型载体将有望实现高效、低毒、靶向特异等特点,从而成为基因治疗中的理想载体。