Secure and effective gene delivery system of plasmid DNA coated by polynucleotide

Polynucleotides are anionic macromolecules which are expected to transfer into the targeted cells through specific uptake mechanisms. So, we developed polynucleotides coating complexes of plasmid DNA (pDNA) and polyethylenimine (PEI) for a secure and efficient gene delivery system and evaluated their usefulness. Polyadenylic acid (polyA), polyuridylic acid (polyU), polycytidylic acid (polyC), and polyguanylic acid (polyG) were examined as the coating materials. pDNA/PEI/polyA, pDNA/PEI/polyU, and pDNA/PEI/polyC complexes formed nanoparticles with a negative surface charge although pDNA/PEI/polyG was aggregated. The pDNA/PEI/polyC complex showed high transgene efficiency in B16-F10 cells although there was little efficiency in pDNA/PEI/polyA and pDNA/PEI/polyU complexes. An inhibition study strongly indicated the specific uptake mechanism of pDNA/PEI/polyC complex. Polynucleotide coating complexes had lower cytotoxicity than pDNA/PEI complex. The pDNA/PEI/polyC complex showed high gene expression selectively in the spleen after intravenous injection into mice. The pDNA/PEI/polyC complex showed no agglutination with erythrocytes and no acute toxicity although these were observed in pDNA/PEI complex. Thus, we developed polynucleotide coating complexes as novel vectors for clinical gene therapy, and the pDNA/PEI/polyC complex as a useful candidate for a gene delivery system.     

非病毒载体介导pBMP-2转染MC3T3-E1细胞的体外成骨分化研究

目的 :探讨非病毒载体Gen Escort TM II介导质粒骨形态发生蛋白2(p BMP-2)转染对MC3T3-E1细胞增殖和体外成骨分化的影响。方法:以非病毒载体Gen Escort TM II介导报告基因质粒增强型绿色荧光蛋白(p EGFP)转染MC3T3-E1细胞,优化转染条件,荧光显微镜和流式细胞仪评价转染效率。p BMP-2转染MC3T3-E1细胞后,MTT法检测细胞增殖情况,染色法检测碱性磷酸酶(ALP)和茜素红S(ARS)的表达,Real-time PCR分析成骨细胞标志基因ALP、骨涎蛋白(BSP)、Runt相关转录因子2(Runx2)、骨钙素(OCN)和骨桥蛋白(OPN)的表达。结果 :Gen Escort TM II介导p EGFP转染MC3T3-E1细胞后,转染效率达35.02±4.42,MTT结果示基因转染对细胞增殖无影响(P>0.05)。p BMP-2转染组的ALP、ARS表达较p EGFP转染组和未转染组显著,同时Real-time PCR检测结果示,转染后6 d,p EGFP转染组和未转染组相比,p BMP-2转染组的成骨基因ALP、BSP、Runx2、OCN、OPN的表达量差异有显著性意义(P<0.05)。结论:非病毒载体Gen Escort TM II介导BMP-2转染MC3T3-E1细胞可诱导其向成骨方向分化。     

The construction of a novel kind of non-viral gene delivery vector based on protein as core backbone

Background and Objectives   A novel kind of non-viral gene delivery vector based on transferrin (Tf) as the core component was constructed with high transfection efficiency and low toxicity.
Materials and Methods   The synthesis vector of Tf-PEI600 was confirmed by different physicochemical methods, including ¹H nuclear magnetic resonance, gel permeation chromatography, X-ray and thermogravimetric analysis. The cytotoxicity and gene delivery efficiency of the synthesized vector were verified by in vitro experiments.
Results   The agarose gel electrophoresis assay indicated that the novel copolymer Tf-PEI600 could efficiently condense plasmid DNA and the condensed nanoparticles exhibited a spherical shape. As the weight ratio of Tf-PEI600 to DNA reached 15·0, the particle size (about 200 nm) and the zeta potential (about 20 mV) of the nanoparticles became optimal for gene delivery. The methylthiazolyl tetrazolium (MTT) assay showed the cytotoxicity of Tf-PEI600 to be similar to that of PEI600 and much lower than that of PEI25kDa. In gene-delivery experiments with COS-7 cells and HepG2 cells, the Tf-PEI600 showed about a 30- to 53-fold higher efficiency than PEI600 and nearly equal to that of PEI25kDa.
Conclusions   These data suggest that Tf-PEI600, with the advantages of low toxicity and high gene-delivery efficiency, might have great prospects in the practice of gene delivery. The core-shell structure of Tf-PEI600 also provided a novel strategy for the construction of non-viral gene delivery vectors.     

Response of Human Osteoblastic and Osteoclastic Cells to AH Plus and Pulp Canal Sealer Containing Quaternary Ammonium Polyethylenimine Nanoparticles

Introduction

The incorporation of quaternary ammonium polyethylenimine (QPEI) nanoparticles into endodontic sealers induces alterations in their structure and surface properties, which may affect the compatibility with the periapical tissues. This work addressed the behavior of human bone cells exposed to extracts from commercial and QPEI containing AH Plus (DeTrey, Konstanz, Germany) and Pulp Canal Sealer EWT (PCS; Kerr Italia Srl, Salerno, Italy).

Methods

Freshly mixed AH Plus and PCS or containing 2% QPEI (0.3 mL spread over the well bottom of a 24-well plate) were extracted with culture medium (1.5 mL for 24 hours at 37°C) and diluted (1:20–1:5000). Osteoblastic or osteoclastic cells were cultured in the presence of QPEI particles (1%–10%) and were exposed to the extracts from unmodified and QPEI containing sealers.

Results

QPEI nanoparticles, at 1% and 2%, did not affect cell behavior. On osteoblastic cells, AH Plus and PCS increased DNA at 1:2500 dilution (levels ≤1:100 were cytotoxic). Alkaline phosphatase activity decreased at dilutions ≤1:500. Comparatively, QPEI containing AH Plus increased DNA at 1:2500 and 1:500 dilutions, and QPEI containing PCS induced ALP activity at 1:2500 and 1:500 dilutions. Regarding osteoclastic cells, DNA increased (AH Plus) or was not affected (PCS) at dilutions up to 1:500 and decreased with more concentrated extracts. Tartrate-resistant acid phosphatase activity decreased with dilutions ≤1:500 for both sealers. QPEI containing sealers presented a similar behavior. The sealers affected some intracellular signaling pathways, and QPEI containing sealers further modulate these mechanisms.

Conclusions

QPEI nanoparticles, at 2%, did not affect cell behavior. However, the incorporation of 2% QPEI particles into AH Plus and PCS modulates the proliferation and differentiation of bone cells, depending on the sealer and the cell type, without increasing the sealers' cytotoxicity.     

聚乙烯亚胺阳离子高聚物对白色念珠菌的体外抗真菌活性

目的:评估阳离子高分子聚合物聚乙烯亚胺(PEI)的潜在抗真菌活性. 方法: 通过体外实验测定不同分子质量PEI对两株白色念珠菌(MY7245和MY7238)的最小抑菌浓度和杀灭速率,确定PEI的体外抗真菌活性. 结果: 白色念珠菌对PEI非常敏感,PEI的体外杀菌活性取决于真菌菌株和聚合物分子质量,大分子质量PEI(25 ku)对于MY7245的最小抑菌浓度是3.13 mg/L,而对MY7238是1.56 mg/L;小分子质量PEI对两株菌的最小抑菌浓度都是1.56 mg/L. 当用低于最小抑菌浓度(0.5×MIC)的PEI处理24 h之后,MY7238的活菌数减少到原来的1/300至1/700;若用二倍最小抑菌浓度(2×MIC)时活菌数则显著减少到原来的1/10 000. 结论: ①PEI的抗真菌活性与文献报道的其他阳离子型多肽药物效果相当. ②首次发现阳离子聚合物可作为杀真菌剂的应用潜力,且具有价格相对低廉、透膜性良好、无免疫原性的优点.     

Partial Acetylation of Polyethylenimine Enhances In Vitro Gene Delivery

PURPOSE: Polyethylenimine (PEI) is a highly effective gene delivery vector, but because it is an off-the shelf material, its properties may not be optimal. To investigate the effects of the protonation properties of the polymer, we generated PEI derivatives by acetylating varying fractions of the primary and secondary amines to form secondary and tertiary amides, respectively. METHODS: Reaction of PEI with increasing amounts of acetic anhydride at 60 degrees C for 4.5 h yielded polymers with 15%, 27%, and 43% of the primary amines modified with acetyl groups. Polymer-DNA complexes were characterized by dynamic light scattering and zeta potential measurements. Cytotoxicity of the polymers was assessed by XTT assay for metabolic activity, and gene delivery efficiency was determined as the relative expression of a luciferase gene in MDA-MB-231 and C2C12 cell lines. RESULTS: Acetylation of PEI decreased the "physiological buffering capacity," defined as the moles of protons absorbed per mole of nitrogen on titration from pH 7.5 to 4.5, from 0.29 mol H+/mol N to 0.17 mol H+/mot N, 0.12 mol H+/mol N, and 0.090 mol H+/mol N for PEI-Ac15, PEI-Ac27, and PEI-Ac43, respectively. In addition, acetylation decreased the zeta potential of polyplexes from 14 mV to 8-11 mV and increased the polyplex diameter by two- to threefold. Surprisingly, acetylation had a negligible effect on cytotoxicity of the polymers and increased gene delivery effectiveness by up to 21-fold compared to unmodified PEI, both in the presence and absence of serum. CONCLUSIONS: Reduction of the buffering capacity of PEI greatly enhanced the gene delivery activity of the polymer. The mechanism is not yet understood, but the enhancement may be caused by more effective polyplex unpackaging, altered endocytic trafficking, and/or increased lipophilicity of acetylated PEI-DNA complexes. Future studies will address these possibilities in more detail.     

聚乙烯亚胺介导NIH-3T3细胞体外转染pEGFP-N1系统优化

目的 优化聚乙烯亚胺(polyethylenimine,PEI)介导的细胞转染以提高目的 基因在细胞中的表达强度.方法 根据PEI/DNA不同的质量比(0:1、0.5:1、1:1、2:1、3:1、4:1、5:1、6:1、7:1、8:1、9:1、10:1)利用凝胶阻滞分析结合的情况;不同质量比PEI/DNA形成的复合物对NIH-3T3细胞的转染通过细胞表达强度获得最佳PEb/DNA质量比;比较实验组l(PEI/DNA质量比2:1)、实验组2(重复PEL/DNA质量比2:1)及实验组3(PEI/DNA质茸比4:2)3种转染方案,探讨重复转染的方法是否提高细胞表达强度.结果 ①通过凝胶阻滞分析PEI/DNA能够稳定结合的质量比是1:1～10:1;②PEI/DNA复合物对NIH-3T3细胞进行转染,其荧光表达强度当PEI/DNA(质量比)≤2时随着PEI的增加而提高,当PEI/DNA(质量比)>2时,荧光表达强度反而降低;③采用重复转染的方法实验组2细胞荧光表达强度(24.08±0.28)%明显高于实验组1(8.97±4.02)%和实验组3(14.24±2.68)%(P<0.05).结论 在PEI/DNA(质量比)=2的条件下,PEI/DNA复合物转化NIH-3T3细胞的效果比较理想,重复转染可以提高细胞转染后荧光表达强度.     

流式细胞仪分选非染色S期CHO细胞有助于提高聚乙烯亚胺的转染效率

目的利用流式细胞仪(flow cytometry,FCM)分选获得非染色、同步化的中国仓鼠卵巢(Chinese hamsterovary,CHO)细胞,观察聚乙烯亚胺(polyethylenimine,PEI)在不同周期CHO细胞的瞬时转染和稳定转染效率。方法悬浮培养CHODG44细胞,应用流式细胞仪的光学参数-前向角和侧向角,在细胞主群中设置12个门,然后进行分选。分选出的各门内细胞经碘化丙啶(PI)染色进行细胞周期检测。以PEI为载体分别向含G0/G1、S、G2/M比例较高的细胞亚群和未经分选的细胞转染含绿荧光蛋白(Green Fluorescence Protein,GFP)质粒,利用流式细胞仪检测瞬时转染、稳定转染效率。结果细胞DNA含量与侧向角相关,各细胞亚群G0/G1期细胞所占比例总体上随着侧向角的增大而减小,G2/M细胞所占比例随着侧向角的增大而增多。低侧向角的细胞亚群P2内G0/G1期细胞比例可达80.49%,中侧向角的细胞亚群P7内S期细胞比例可达61.14%,高侧向角的细胞亚群P13内G2/M期细胞比例可达49.88%。转染48h,GFP阳性细胞在G2/M期细胞比例较高的细胞亚群所占比例最高,但瞬时转染效率,S期细胞比例较高的细胞群大于G2/M期细胞比例较高的细胞群。转染2w,稳定转染效率仍为S期细胞比例较高的细胞亚群大于其它细胞亚群及未分选的混合细胞群。结论应用流式细胞仪的光学参数可以有效分选非诱导、无染色分别含有较高比例各周期时相的CHO细胞;PEI的转染效率与转染时细胞所处的细胞周期时相相关,且S期细胞亚群的瞬时和稳定转染效率均高于其它细胞周期亚群。     

A novel polymer-lipid hybrid nanoparticle for efficient nonviral gene delivery

Aim:

To develop a novel non-viral vector with high transfection efficiency and low cytotoxicity.

Methods:

Poly (ethylene glycol)-distearoylphosphatidylethanolamine (PEG-DSPE) was incorporated into polymer-lipid hybrid nanoparticles (PLN) to construct a PEG-DSPE modified long circulating PLN (L-PLN). The L-PLN was prepared by the emulsifying-solvent evaporation method, L-PLN and L-PLN/DNA complexes were characterized. Both HEK293 and MDA-MB-231 cells transfected by L-PLN/DNA complexes were observed under a fluorescence microscope. The transfection efficiency of the complexes to HEK293 cells was further evaluated by flow cytometry.

Results:

The GFP fluorescence intensity in HEK293 cells transfected by the L-PLN/DNA complexes (N/P=10) was about 37.2%, which was higher than those transfected by PLN alone or commercial Lipofectamine^TM 2000. The L-PLN exhibited minimal toxicity at a low N/P ratio compared with other vectors.

Conclusion:

L-PLN as a novel gene delivery system, has higher transfection efficiency and acceptable cytotoxicity compared to the corresponding PLN, which is beneficial for the development of non-viral gene transfer vectors and may offer an alternative strategy for the future gene therapy.     

In vivo protein transduction to the CNS

Proteins and peptides are useful research and therapeutic tools, however applications are limited because delivery to the desired location is not easily achievable. There are two hurdles in protein/peptide delivery to the brain: the blood-brain barrier and intracellular penetration. Penetration to both brain and the intracellular space can be achieved by adjusting hydrophilicity, and small molecule pharmacological agents have been successfully developed using this approach. But with proteins and peptides, it is difficult to modify the hydrophilicity without influencing biological functions. Trans-acting factor protein from the human immunodeficiency virus contains a highly conserved cationic peptide sequence necessary for transduction across the cell membrane. While trans-acting factor peptide has been used for in vitro protein transduction, its in vivo application is very limited because it is rapidly degraded by proteolysis. Polyethylenimine is a chemically synthesized small molecule cationization agent; the charge density is greater than a peptide-based cationic cluster such as trans-acting factor, and it is resistant to proteolysis in vivo. We first tested intracellular protein transduction following direct brain injection in mice using polyethylenimine-conjugated green fluorescence protein and beta-galactosidase (molecular weights 29 and 540 kDa, respectively). Polyethylenimine-conjugates penetrated to the intracellular space immediately surrounding the injection site within one hour. We further tested polyethylenimine-mediated protein transduction following intranasal administration, which bypasses the blood-brain barrier. Polyethylenimine-conjugates in pH 7.5 solution did not reach the brain, probably because the polyethylenimine-conjugates penetrated into the intracellular space where first exposed to the tissue, i.e. at the nasal mucosae. We temporarily reduced the electrostatic interaction between cationized polyethylenimine-conjugates and cellular surfaces by adjusting the pH to 4.5; solution rapidly reached the brain and penetrated to the intracellular space. This study suggests that polyethylenimine is a useful protein transduction agent in the brain in vivo, and adjusting cationic charge interaction can determine the extent of brain penetration.